Polynucleotide compositions encoding Cry1Ac/Cry1F chimeric O-endotoxins

ABSTRACT

Disclosed are novel synthetically-modified  B. thuringiensis  chimeric crystal proteins having improved insecticidal activity against coleopteran, dipteran and lepidopteran insects. Also disclosed are the nucleic acid segments encoding these novel peptides. Methods of making and using these genes and proteins are disclosed as well as methods for the recombinant expression, and transformation of suitable host cells. Transformed host cells and transgenic plants expressing the modified endotoxin are also aspects of the invention.

The present application is a divisional of U.S. application Ser. No.08/754,490, filed Nov. 20, 1996, now U.S. Pat. No. 6.017,534. The entirecontents of which is specifically incorporated herein by reference inits entirety.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of molecularbiology. More particularly, certain embodiments concern novel nucleicacid segments, and genetically-engineered recombinant δ-endotoxinsderived from Bacillus thuringiensis. More particularly, it concernsnovel chimeric crystal proteins and the chimeric cry gene segments whichencode them. Various methods for making and using these DNA segments,methods of producing the encoded proteins, methods for makingsynthetically-modified chimeric crystal proteins, and methods of makingand using the synthetic crystal proteins are disclosed, such as, forexample, the use of nucleic acid segments as diagnostic probes andtemplates for protein production, and the use of proteins, fusionprotein carriers and peptides in various immunological and diagnosticapplications. Also disclosed is the use of these cry gene fusions andchimeric Cry proteins in the development of transgenic plants whichexpress broad-spectrum insecticidal activity against a variety ofcoleopteran, dipteran, and lepidopteran insects.

1.2 Description of Related Art

1.2.1 Bacillus thuringiensis Crystal Proteins

The Gram-positive soil bacterium Bacillus thuringiensis is well knownfor its production of proteinaceous parasporal crystals, orδ-endotoxins, that are toxic to a variety of lepidopteran, coleopteran,and dipteran larvae. B. thuringiensis produces crystal proteins duringsporulation which are specifically toxic to certain species of insects.Many different strains of B. thuringiensis have been shown to produceinsecticidal crystal proteins, and compositions comprising B.thuringiensis strains which produce proteins having insecticidalactivity have been used commercially as environmentally-acceptableinsecticides because of their toxicity to the specific target insect,and non-toxicity to plants and other non-targeted organisms.

Commercial formulations of naturally occurring B. thuringiensis isolateshave long been used for the biological control of agricultural insectpests. In commercial production, the spores and crystals obtained fromthe fermentation process are concentrated and formulated for foliarapplication according to conventional agricultural practices.

1.2.2 Nomenclature of Crystal Proteins

A review by Höfte et al., (1989) describes the general state of the artwith respect to the majority of insecticidal B. thuringiensis strainsthat have been identified which are active against insects of the OrderLepidoptera, i.e., caterpillar insects. This treatise also describes B.thuringiensis strains having insecticidal activity against insects ofthe Orders Diptera (ie. flies and mosquitoes) and Coleoptera (i.e.beetles). A number of genes encoding crystal proteins have been clonedfrom several strains of B. thuringiensis. Höfte et al. (1989) discussesthe genes and proteins that were identified in B. thuringiensis prior to1990, and sets forth the nomenclature and classification scheme whichhas traditionally been applied to B. thuringiensis genes and proteins.cry1 genes encode lepidopterantoxic Cry1 proteins. cry2 genes encodeCry2 proteins that are toxic to both lepidopterans and dipterans. cry3genes encode coleopteran-toxic Cry3 proteins, while cry4 genes encodedipteran-toxic Cry4 proteins, etc.

Recently a new nomenclature has been proposed which systematicallyclassifies the Cry proteins based upon arnino acid sequence homologyrather than upon insect target specificities. This classification schemeis summarized in TABLE 1.

TABLE 1 Revised B. thuringiensis δ-Endotoxin Nomenclature^(a) New OldGenBank Accession # Cry1Aa CryIA(a) M11250 Cry1Ab CryIA(b) M13898 Cry1AcCryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1BaCryIB X06711 Cry1Bb ETS L32020 Cry1Bc PEG5 Z46442 Cry1Ca CryIC X07518Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1Db PrtB Z22511 Cry1EaCryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIF M63897 Cry1Fb PrtDZ22512 Cry1G PrtA Z22510 Cry1H PrtC Z22513 Cry1Hb U35780 Cry2a CryVX62821 Cry2b CryV U07642 Cry2Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1KU28801 Cry2Aa CryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252Cry3A CryIIIA M22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3CCryIIID X59797 Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a)L07025 Cry5Ab CryVA(b) L07026 Cry5B U19725 Cry6A CryVIA L07022 Cry6BCryVIB L07024 Cry7Aa CryIIIC M64478 Cry7Ab CryIIICb U04367 Cry8A CryIIIEU04364 Cry8B CryIIIG U04365 Cry8C CryIIIF U04366 Cry9A CryIG X58120Cry9B CryIX X75019 Cry9C CryIH Z37527 Cry10A CryIVC M12662 Cry11A CryIVDM31737 Cry11B Jeg80 X86902 Cry12A CryVB L07027 Cry13A CryVC L07023Cry14A CryVD U13955 Cry15A 34kDa M76442 Cry16A cbm71 X94146 Cyt1A CytAX03182 Cyt2A CytB Z14147 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

1.2.3 Mode of Crystal Protein Toxicity

All δ-endotoxin crystals are toxic to insect larvae by ingestion.Solubilization of the crystal in the midgut of the insect releases theprotoxin form of the δ-endotoxin which, in most instances, issubsequently processed to an active toxin by midgut protease. Theactivated toxins recognize and bind to the brush-border of the insectmidgut epithelium through receptor proteins. Several putative crystalprotein receptors have been isolated from certain insect larvae (Knightet al., 1995; Gill et al., 1995; Masson et al., 1995). The binding ofactive toxins is followed by intercalation and aggregation of toxinmolecules to form pores within the midgut epithelium. This process leadsto osmotic imbalance, swelling, lysis of the cells lining the midgutepithelium, and eventual larvae mortality.

1.2.4 Molecular Biology of δ-Endotoxins

With the advent of molecular genetic techniques, various δ-endotoxingenes have been isolated and their DNA sequences determined. These geneshave been used to construct certain genetically engineered B.thuringiensis products that have been approved for commercial use.Recent developments have seen new δ-endotoxin delivery systemsdeveloped, including plants that contain and express geneticallyengineered δ-endotoxin genes.

The cloning and sequencing of a number of δ-endotoxin genes from avariety of Bacillus thuringiensis strains have been described and aresummarized by Höfte and Whiteley, 1989. Plasmid shuttle vectors designedfor the cloning and expression of δ-endotoxin genes in E. coli or B.thuringiensis are described by Gawron-Burke and Baum (1991). U.S. Pat.No. 5,441,884 discloses a site-specific recombination system forconstructing recombinant B. thuringiensis strains containing δ-endotoxingenes that are free of DNA not native to B. thuringiensis.

The Cry1 family of crystal proteins, which are primarily active againstlepidopteran pests, are the best studied class of δ-endotoxins. Thepro-toxin form of Cry1 δ-endotoxins consist of two approximately equalsized segments. The carboxyl-half, or pro-toxin segment, is not toxicand is thought to be important for crystal formation (Arvidson et al.,1989). The amino-half of the protoxin comprises the active-toxin segmentof the Cry1 molecule and may be further divided into three structuraldomains as determined by the recently described crystallographicstructure for the active toxin segment of the Cry1Aa δ-endotoxin(Grochulski et al., 1995). Domain 1 occupies the first third of theactive toxin and is essential for channel formation (Thompson et al.,1995). Domain 2 and domain 3 occupy the middle and last third of theactive toxin, respectively. Both domains 2 and 3 have been implicated inreceptor binding and insect specificity, depending on the insect andδ-endotoxin being examined (Thompson et al., 1995).

1.2.5 Chimeric Crystal Proteins

In recent years, researchers have focused effort on the construction ofhybrid δ-endotoxins with the hope of producing proteins with enhancedactivity or improved properties. Advances in the art of moleculargenetics over the past decade have facilitated a logical and orderlyapproach to engineering proteins with improved properties. Site-specificand random mutagenesis methods, the advent of polymerase chain reaction(PCR™) methodologies, and the development of recombinant methods forgenerating gene fusions and constructing chimeric proteins havefacilitated an assortment of methods for changing amino acid sequencesof proteins, fusing portions of two or more proteins together in asingle recombinant protein, and altering genetic sequences that encodeproteins of commercial interest.

Unfortunately, for crystal proteins, these techniques have only beenexploited in limited fashion. The likelihood of arbitrarily creating achimeric protein with enhanced properties from portions of the numerousnative proteins which have been identified is remote given the complexnature of protein structure, folding, oligomerization, activation, andcorrect processing of the chimeric protoxin to an active moiety. Only bycareful selection of specific target regions within each protein, andsubsequent protein engineering can toxins be synthesized which haveimproved insecticidal activity.

Some success in the area, however, has been reported in the literature.For example, the construction of a few hybrid δ-endotoxins is reportedin the following related art: Intl. Pat. Appl. Publ. No. WO 95/30753discloses the construction of hybrid B. thuringiensis δ-endotoxins forproduction in Pseudomonas fluorescens in which the non-toxic protoxinfragment of Cry1F has been replaced by the non-toxic protoxin fragmentfrom the Cry1Ac/Cry1Ab that is disclosed in U.S. Pat. No. 5,128,130.

U.S. Pat. No. 5,128,130 discloses the construction of hybrid B.thuringiensis δ-endotoxins for production in P. fluorescens in which aportion of the non-toxic protoxin segment of Cry1Ac is replaced with thecorresponding non-toxic protoxin fragment of Cry1Ab. U.S. Pat. No.5,055,294 discloses the construction of a specific hybrid δ-endotoxinbetween Cry1Ac (amino acid residues 1-466) and Cry1Ab (amino acidresidues 466-1155) for production in P. fluorescens. Although theaforementioned patent discloses the construction of a hybrid toxinwithin the active toxin segment, no specifics are presented in regard tothe hybrid toxin's insecticidal activity. Intl. Pat. Appi. Publ. No. WO95/30752 discloses the construction of hybrid B. thuringiensisδ-endotoxins for production in P. fluorescens in which the non-toxicprotoxin segment of Cry1C is replaced by the non-toxic protoxin segmentfrom Cry1Ab. The aforementioned application further discloses that theactivity against Spodoptera exigua for the hybrid δ-endotoxin isimproved over that of the parent active toxin, Cry1C.

Intl. Pat. Appl. Publ. No. WO 95/06730 discloses the construction of ahybrid B. thuringiensis δ-endotoxin consisting of domains 1 and 2 ofCry1E coupled to domain 3 and the non-toxic protoxin segment of Cry1C.Insect bioassays performed against Manduca sexta (sensitive to Cry1C andCry1E), Spodoptera exigua (sensitive to Cry1C), and Mamestra brassicae(sensitive to Cry1C) show that the hybrid Cry1E/Cry1C hybrid toxin isactive against M. sexta, S. exigua, and M. brassicae. The bioassayresults were expressed as EC₅₀ values (toxin concentration giving a 50%growth reduction) rather than LC₅₀ values (toxin concentration giving50% mortality). Although the δ-endotoxins used for bioassay wereproduced in B. thuringiensis, only artificially-generated activesegments of the δ-endotoxins were used, not the naturally-producedcrystals typically produced by B. thuringiensis that are present incommercial B. thuringiensis formulations. Bioassay results indicatedthat the LC₅₀ values for the hybrid Cry1E/Cry1C crystal against S.frugiperda were 1.5 to 1.7 fold lower (more active) than for nativeCry1C. This art also discloses the construction of a hybrid B.thuringiensis δ-endotoxin between Cry1Ab (domains 1 and 2) and Cry1C(domain 3 and the non-toxic protoxin segment), although no data aregiven regarding the hybrid toxin's activity or usefulness.

Lee et al. (1995) report the construction of hybrid B. thuringiensisδ-endotoxins between Cry1Ac and Cry1Aa within the active toxin segment.Artificially generated active segments of the hybrid toxins were used toexamine protein interactions in susceptible insect brush bordermembranes vesicles (BBMV). The bioactivity of the hybrid toxins was notreported.

Honee et al. (1991) report the construction of hybrid δ-endotoxinsbetween Cry1C (domain 1) and Cry1Ab (domains 2 and 3) and the reciprocalhybrid between Cry1Ab (domain 1) and Cry1C (domains 2 and 3). Thesehybrids failed to show any significant increase in activity againstsusceptible insects. Furthermore, the Cry1C (domain 1)/Cry1Ab (domains 2and 3) hybrid toxin was found to be hypersensitive to proteasedegradation. A report by Schnepf et al. (1990) discloses theconstruction of Cry1Ac hybrid toxin in which a small portion of domain 2was replaced by the corresponding region of Cry1Aa, although nosignificant increase in activity against susceptible insect larvae wasobserved.

1.3 Deficiencies in the Prior Art

The limited successes in producing chimeric crystal proteins which haveimproved activity have negatively impacted the field by thwartingefforts to produce recombinantly-engineered crystal protein forcommercial development, and to extend the toxic properties and hostspecificities of the known endotoxins. Therefore, what is lacking in theprior art are reliable methods and compositions comprisingrecombinantly-engineered crystal proteins which have improvedinsecticidal activity, broad-host-range specificities, and which aresuitable for commercial production in Bacillus thuringiensis.

2. SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations in the priorart by providing novel chimeric δ-endotoxins which have improvedinsecticidal properties, and broad-range specificities.

Disclosed are methods for the construction of B. thuringiensis hybridδ-endotoxins comprising amino acid sequences from native Cry1Ac andCry1F crystal proteins. These hybrid proteins, in which all or a portionof Cry1Ac domain 2, all or a portion of Cry1Ac domain 3, and all or aportion of the Cry1Ac protoxin segment is replaced by the correspondingportions of Cry1F, possess not only the insecticidal characteristics ofthe parent δ-endotoxins, but also have the unexpected and remarkableproperties of enhanced broad-range specificity which is not proficientlydisplayed by either of the native δ-endotoxins from which the chimericproteins were engineered.

Specifically, the present invention discloses and claimsgenetically-engineered hybrid δ-endotoxins which comprise a portion of aCry1Ac crystal protein fused to a portion of a Cry1F crystal protein.These chimeric endotoxins have broad-range specificity for the insectpests described herein.

In a further embodiment, the present invention also discloses and claimsrecombinant B. thuringiensis hybrid δ-endotoxins which comprise aportion of Cry1Ab, Cry1F, and Cry1Ac in which all or a portion of Cry1Abdomain 2 or all or a portion of Cry1Ab domain 3 is replaced by thecorresponding portions of Cry1F and all or a portion of the Cry1Abprotoxin segment is replaced by the corresponding portions of Cry1Ac.Exemplary hybrid δ-endotoxins between Cry1Ab and Cry1F are identified inSEQ ID NO:13 and SEQ ID NO:14.

One aspect of the present invention demonstrates the unexpected resultthat certain hybrid δ-endotoxins derived from Cry1Ac and Cry1F proteinsexhibit not only the insecticidal characteristics of the parentδ-endotoxins, but also possess insecticidal activity which is notproficiently displayed by either of the parent δ-endotoxins.

Another aspect of the invention further demonstrates the unexpectedresult that certain chimeric Cry1Ab/Cry1F proteins maintain not only theinsecticidal characteristics of the parent δ-endotoxins, but alsoexhibit insecticidal activity which is not displayed by either thenative Cry1Ab or Cry1F endotoxins.

The present invention also encompasses Cry1Ac/Cry1F and Cry1Ab/Cry1Fhybrid δ-endotoxins that maintain the desirable characteristics neededfor commercial production in B. thuringiensis. Specifically, the hybridδ-endotoxins identified in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:26, SEQ ID NO:28, and SEQ ID NO:30 can efficiently formproteinaceous parasporal inclusions in B. thuringiensis and have thefavorable characteristics of solubility, protease susceptibility, andinsecticidal activity of the parent δ-endotoxins.

In a further embodiment, the present invention also discloses and claimsrecombinant B. thuringiensis hybrid δ-endotoxins which comprise aportion of Cry1Ac and Cry1C in which all or a portion of Cry1Ac domain 3is replaced by the corresponding portions of Cry1C and all or a portionof the Cry1Ac protoxin segment is replaced by the corresponding portionof Cry1C. Exemplary hybrid δ-endotoxins between Cry1Ac and Cry1C areidentified in SEQ ID NO:29 and SEQ ID NO:30.

One aspect of the present invention demonstrates the unexpected resultthat, although neither Cry1Ac nor Cry1C possess S. frugiperda activity,the Cry1Ac/Cry1C hybrid δ-endotoxin identified by SEQ ID NO:29 and SEQID NO:30 has significant activity against S. frugiperda. Furthermore,the Cry1Ac/Cry1C hybrid δ-endotoxin identified by SEQ ID NO:29 and SEQID NO:30 has significantly better activity against S. exigua than theCry1C parental δ-endotoxin.

The present invention further pertains to the recombinant nucleic acidsequences which encode the novel crystal proteins disclosed herein.Specifically, the invention discloses and claims the nucleic acidsequences of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQID NO:27, and SEQ ID NO:29; nucleic acid sequences which arecomplementary to the nucleic acid sequences of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29; andnucleic acid sequences which hybridize to the sequences of SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, and SEQ IDNO:29.

The novel hybrid δ-endotoxins disclosed herein are useful in the controlof a broad range of insect pests. These hybrid δ-endotoxins aredescribed in FIG. 1 and are disclosed in SEQ ID NO:10, SEQ ID NO:12, SEQID NO: 14, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30. The nucleicacid segments encoding these proteins are disclosed in SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29.The insecticidal and biochemical properties of the hybrid δ-endotoxinsare described by FIG. 2, FIG. 3, and TABLE 3, TABLE 4, TABLE 5, andTABLE 6. The broad host range of the improved δ-endotoxins specified inthe present invention is useful in circumventing dilution effects causedby expressing multiple δ-endotoxin genes within a single B.thuringiensis strain. Expression of such a broad host range δ-endotoxinin plants is expected to impart protection against a wider variety ofinsect pests.

The impetus for constructing these and other hybrid δ-endotoxins is tocreate novel toxins with improved insecticidal activity, increasedhost-range specificity, and improved production characteristics. The DNAsequences listed in TABLE 5 define the exchange points for the hybridδ-endotoxins pertinent to the present invention and as oligonucleotideprimers, may be used to identify like or similar hybrid δ-endotoxins bySouthern or colony hybridization under conditions of moderate to highstringency. Researchers skilled in the art will recognize the importanceof the exchange site chosen between two or more δ-endotoxins can beachieved using a number of in vivo or in vitro molecular genetictechniques. Small variations in the exchange region between two or moreδ-endotoxins may yield similar results or, as demonstrated for EG11062and EG11063, adversely affect desirable traits. Similarly, largevariations in the exchange region between two or more δ-endotoxins mayhave no effect on desired traits, as demonstrated by EG11063 andEG11074, or may adversely affect desirable traits, as demonstrated byEG11060 and EG11063.

Favorable traits with regard to improved insecticidal activity,increased host range, and improved production characteristics may beachieved by other such hybrid δ-endotoxins including, but not limitedto, the cry1, cry2, cry3, cry4, cry5, cry6, cry7, cry8, cry9, cry10,cry11, cry12, cry13, cry14, cry15 class of δ-endotoxin genes and the B.thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes ofB. thuringiensis insecticidal proteins include, but are not limited toCry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba, Cry1Bb, Cry1Ca, Cry1Cb,Cry1Da, Cry1Db, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Ha, Cry2a,Cry2b, Cry1Ja, Cry1Ka, Cry11Aa, Cry11Ab, Cry12Aa, Cry3Ba, Cry3Bb, Cry3C,Cry4a, Cry4Ba, Cry5a, Cry5Ab, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry8Aa,Cry8Ba, Cry8Ca, Cry9Aa, Cry9Ba, Cry9Ca, Cry10Aa, Cry11Aa, Cry12Aa,Cry13Aa, Cry14Aa, Cry15Aa, Cyt1Aa, and Cyt2Aa. Related hybridδ-endotoxins would consist of the arnino portion of one of theaforementioned δ-endotoxins, including all or part of domain 1 or domain2, fused to all or part of domain 3 from another of the aforementionedδ-endotoxins. The non-active protoxin fragment of such hybridδ-endotoxins may consist of the protoxin fragment from any of theaforementioned δ-endotoxins which may act to stabilize the hybridδ-endotoxin as demonstrated by EG11087 and EG11091 (see e.g., TABLE 3).Hybrid δ-endotoxins possessing similar traits as those described in thepresent invention could be constructed by conservative, or “similar”replacements of amino acids within hybrid δ-endotoxins. Suchsubstitutions would mimic the biochemical and biophysical properties ofthe native amino acid at any position in the protein. Amino acidsconsidered similar include for example, but are not limited to:

Ala, Ser, and Thr

Asp and Glu

Asn and Gln

Lys and Arg

Ile, Leu, Met, and Val

Phe, Tyr, and Trp

Researchers skilled in the art will recognize that improved insecticidalactivity, increased host range, and improved production characteristicsimparted upon hybrid δ-endotoxins may be further improved by alteringthe genetic code for one or more amino acid positions in the hybridδ-endotoxin such that the position, or positions, is replaced by anyother amino acid. This may be accomplished by targeting a region orregions of the protein for mutagenesis by any number of establishedmutagenic techniques, including those procedures relevant to the presentinvention. Such techniques include site-specific mutagenesis (Kunkle,1985; Kunkle et al., 1987), DNA shuffling (Stemmer, 1994), and PCR™overlap extension (Horton et al., 1989). Since amino acids situated ator near the surface of a protein are likely responsible for itsinteraction with other proteinaceous or non-proteinaceous moieties, theymay serve as “target” regions for mutagenesis. Such surface exposedregions may consist of, but not be limited to, surface exposed aminoacid residues within the active toxin fragment of the protein andinclude the inter-α-helical or inter-β-strand “loop” -regions ofδ-endotoxins that separate α-helices within domain 1 and β-strandswithin domain 2 and domain 3. Such procedures may favorably change theprotein's biochemical and biophysical characteristics or its mode ofaction as outlined in the Section 1. These include, but are not limitedto: 1) improved crystal formation, 2) improved protein stability orreduced protease degradation, 3) improved insect membrane receptorrecognition and binding, 4) improved oligomerization or channelformation in the insect midgut endothelium, and 5) improved insecticidalactivity or insecticidal specificity due to any or all of the reasonsstated above.

2.1 Crystal Protein Transgenes and Transgenic Plants

In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel chimeric crystal proteins of the present invention.The process of producing transgenic plants is well-known in the art. Ingeneral, the method comprises transforming a suitable host cell with aDNA segment which contains a promoter operatively linked to a codingregion that encodes a B. thuringiensis Cry1Ac-1F or Cry1Ab-Cry1F, or aCry1Ab-1Ac-1F chimeric crystal protein. Such a coding region isgenerally operatively linked to a transcription-terminating region,whereby the promoter is capable of driving the transcription of thecoding region in the cell, and hence providing the cell the ability toproduce the recombinant protein in vivo. Alternatively, in instanceswhere it is desirable to control, regulate, or decrease the amount of aparticular recombinant crystal protein expressed in a particulartransgenic cell, the invention also provides for the expression ofcrystal protein antisense mRNA. The use of antisense mRNA as a means ofcontrolling or decreasing the amount of a given protein of interest in acell is well-known in the art.

Another aspect of the invention comprises a transgenic plant whichexpress a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression. The construction and expression of synthetic B.thuringiensis genes in plants has been described in detail in U.S. Pat.No. 5,500,365 and U.S. Pat. No. 5,380,831 (each specificallyincorporated herein by reference).

It is contemplated that in some instances the genome of a transgenicplant of the present invention will have been augmented through thestable introduction of one or more cry1Ac-IF, cry1Ab-IF, orcry1Ab-1Ac-1F transgenes, either native, synthetically-modified, orfurther mutated. In some instances, more than one transgene will beincorporated into the genome of the transformed host plant cell. Such isthe case when more than one crystal protein-encoding DNA segment isincorporated into the genome of such a plant. In certain situations, itmay be desirable to have one, two, three, four, or even more B.thuringiensis crystal proteins (either native orrecombinantly-engineered) incorporated and stably expressed in thetransformed transgenic plant.

A preferred gene which may be introduced includes, for example, acrystal protein-encoding a DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of a transgeniccell line are well-known in the art, and are discussed herein. Vectors,plasmids, cosmids, yeast artificial chromosomes (YACs) and nucleic acidsegments for use in transforming such cells will, of course, generallycomprise either the operons, genes, or gene-derived sequences of thepresent invention, either native, or synthetically-derived, andparticularly those encoding the disclosed crystal proteins. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even gene sequences which have positively- ornegatively-regulating activity upon the particular genes of interest asdesired. The DNA segment or gene may encode either a native or modifiedcrystal protein, which will be expressed in the resultant recombinantcells, and/or which will impart an improved phenotype to the regeneratedplant. Nucleic acid sequences optimized for expression in plants havebeen disclosed in Intl. Pat. Appl. Publ. No. WO 93/07278 (specificallyincorporated herein by reference).

Such transgenic plants may be desirable for increasing the insecticidalresistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a transgenic DNA segment encodingCry1Ac-1F and/or Cry1Ab-1F and/or Cry1Ab-1Ac-1F crystal protein(s) whichpossess broad insects. Particularly preferred plants such as grains,including but not limited to corn, wheat, oats, rice, maize, and barley;cotton; soybeans and other legumes; trees, including but not limited toornamental, shrub, fruit, and nut; vegetables, turf and pasture grasses,fruits, berries, citrus, other crops of commercial interest; includinggarden and houseplants.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have astably crystal protein transgene stably incorporated into its genome,and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more chimeric crystal proteins or polypeptidesare aspects of this invention.

2.2 Crystal Protein Screening and Immunodetection Kits

The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides. Saidkit can contain a nucleic acid segment or an antibody of the presentinvention. The kit can contain reagents for detecting an interactionbetween a sample and a nucleic acid or antibody of the presentinvention. The provided reagent can be radio-, fluorescently- orenzymatically-labeled. The kit can contain a known radiolabeled agentcapable of binding or interacting with a nucleic acid or antibody of thepresent invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatograph media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, inmunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.3 ELISAs and Immunoprecipitation

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating crystal protein antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hours, at temperatures preferably on theorder of about 25° to about 27° C. Following incubation, the antiseracontacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

The anti-crystal protein antibodies of the present invention areparticularly useful for the isolation of other crystal protein antigensby immunoprecipitation. Immunoprecipitation involves the separation ofthe target antigen component from a complex mixture, and is used todiscriminate or isolate minute amounts of protein. For the isolation ofmembrane proteins cells must be solubilized into detergent micelles.Nonionic salts are preferred, since other agents such as bile salts,precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

2.4 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areirnmunoglobulins (precluding the use of immunoglobulins bindingbacterial cell wall components), the antigens studied cross-react withthe detecting agent, or they migrate at the same relative molecularweight as a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

2.5 Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies. In particular, the invention concerns epitopic coresequences derived from Cry proteins or peptides.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a crystal protein or polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the crystal protein orpolypeptide will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

The identification of Cry immunodominant epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that shorter antigenic crystal protein-derived peptides willprovide advantages in certain circumstances, for example, in thepreparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to crystal proteins,and in particular Cry and Cry-related sequences. These epitopic coresequences are identified herein in particular aspects as hydrophilicregions of the particular polypeptide antigen. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation, and, hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the crystal protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include. an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

2.6 Nucleic Acid Segments Encoding Crystal Protein Chimeras

The present invention also concerns DNA segments, both native,synthetic, and mutagenized, that can be synthesized, or isolated fromvirtually any source, that are free from total genomic DNA and thatencode the novel chimeric peptides disclosed herein. DNA segmentsencoding these peptide species may prove to encode proteins,polypeptides, subunits, functional domains, and the like of crystalprotein-related or other non-related gene products. In addition theseDNA segments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a crystal protein or peptide refers toa DNA segment that contains crystal protein coding sequences yet isisolated away from, or purified free from, total genomic DNA of thespecies from which the DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species of Bacillus known as B. thuringiensis.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified crystalprotein-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences, operonsequences and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a gene encoding a bacterial crystalprotein, forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a Crypeptide species that includes within its amino acid sequence an aminoacid sequence essentially as set forth in SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.

The term “a sequence essentially as set forth in SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30” meansthat the sequence substantially corresponds to a portion of the sequenceof either SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30 and has relatively few amino acids that are notidentical to, or a biologically functional equivalent of, the aminoacids of any of these sequences. The term “biologically functionalequivalent” is well understood in the art and is further defined indetail herein (e.g., see Illustrative Embodiments). Accordingly,sequences that have between about 70% and about 80%, or more preferablybetween about 81% and about 90%, or even more preferably between about91% and about 99% amino acid sequence identity or functional equivalenceto the amino acids of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30 will be sequences that are“essentially as set forth in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding either of the peptidesequences disclosed in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30, or that are identical to orcomplementary to DNA sequences which encode any of the peptidesdisclosed in SEQ ID NO:10, SEQ ID NO:12 SEQ ID NO:14, SEQ ID NO:26, SEQID NO:28, or SEQ ID NO:30, and particularly those DNA segments disclosedin SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27,or SEQ ID NO:29. For example, DNA sequences such as about 14nucleotides, and that are up to about 10,000, about 5,000, about 3,000,about 2,000, about 1,000, about 500, about 200, about 100, about 50, andabout 14 base pairs in length (including all intermediate lengths) arealso contemplated to be useful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 14, 15,16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; and up to and including sequences of about10,000 nucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequences of SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30,including those DNA sequences which are particularly disclosed in SEQ IDNO:9, SEQ ID NO:11 SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27 or SEQ IDNO:29. Recombinant vectors and isolated DNA segments may thereforevariously include the peptide-coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includethese peptide-coding regions or may encode biologically functionalequivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.7 Recombinant Vectors and Protein Expression

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al., 1989. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Appropriate promoter systemscontemplated for use in highlevel expression include, but are notlimited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise contiguous aminoacid sequences from SEQ ID NO:10, SEQ ID NO:12 SEQ ID NO:14, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30; or any peptide epitope encoded bythe nucleic acid sequences of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29.

Methods for the recombinant expression of crystal proteins and vectorsuseful in the expression of DNA constructs encoding crystal proteins aredescribed in Intl. Pat. Appl. Publ. No. WO 95/02058, specificallyincorporated herein by reference.

2.8 DNA Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelong contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, or SEQ IDNO:29 will find particular utility. Also, nucleic acid segments whichencode at least a 6 amino acid contiguous sequence from SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30,are also preferred. Longer contiguous identical or complementarysequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000,2000, 5000, 10000 etc. (including all intermediate lengths and up to andincluding full-length sequences will also be of use in certainembodiments.

The ability of such nucleic acid probes to specifically hybridize tocrystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so, identical or complementary to DNA sequences of SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, or SEQ IDNO:29, are particularly contemplated as hybridization probes for use in,e.g., Southern and Northern blotting. Smaller fragments will generallyfind use in hybridization embodiments, wherein the length of thecontiguous complementary region may be varied, such as between about10-14 and about 100 or 200 nucleotides, but larger contiguouscomplementarity stretches may be used, according to the lengthcomplementary sequences one wishes to detect.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each specifically incorporated herein by reference), byintroducing selected sequences into recombinant vectors for recombinantproduction, and by other recombinant DNA techniques generally known tothose of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating crystalprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each specificallyincorporated herein by reference) are exemplary of the methods ofhybridization analyses. Teachings such as those found in the texts ofMaloy et al., 1994; Segal 1976; Prokop, 1991; and Kuby, 1994, areparticularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.9 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule In particular embodiments of theinvention, mutated crystal proteins are contemplated to be useful forincreasing the insecticidal activity of the protein, and consequentlyincreasing the insecticidal activity and/or expression of therecombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in TABLE 2.

TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

2.10 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment. in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

2.11 Crystal Protein Compositions as Insecticides and Methods of Use

The inventors contemplate that the chimeric crystal protein compositionsdisclosed herein will find particular utility as insecticides fortopical and/or systemic application to field crops, grasses, fruits andvegetables, and ornamental plants. In a preferred embodiment, thebioinsecticide composition comprises an oil flowable suspension ofbacterial cells which expresses a novel crystal protein disclosedherein. Preferably the cells are B. thuringiensis cells, however, anysuch bacterial host cell expressing the novel nucleic acid segmentsdisclosed herein and producing a crystal protein is contemplated to beuseful, such as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells are B. thuringiensis cells, however, bacteriasuch as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or collodial concentrate.This powder comprises bacterial cells which expresses a novel crystalprotein disclosed herein. Preferred bacterial cells are B. thuringiensiscells, however, bacteria such as B. megaterium, B. subtilis, E. coli, orPseudomonas spp. cells transformed with a DNA segment disclosed hereinand expressing the crystal protein are also contemplated to be useful.Such dry forms of the insecticidal compositions may be formulated todissolve immediately upon wetting, or alternatively, dissolve in acontrolled-release, sustained-release, or other time-dependent manner.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the crystal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the crystal protein gene(s) may be grown in anyconvenient mutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stablizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include Theologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel chimeric Cry proteins may be prepared byrecombinant bacterial expression systems in vitro and isolated forsubsequent field application. Such protein may be either in crude celllysates, suspensions, colloids, etc., or alternatively may be purified,refined, buffered, and/or further processed, before formulating in anactive biocidal formulation. Likewise, under certain circumstances, itmay be desirable to isolate crystals and/or spores from bacterialcultures expressing the crystal protein and apply solutions,suspensions, or collodial preparations of such crystals and/or spores asthe active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, dessicated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran insect, typically onto the foliageof the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular enviromnentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 0.5% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 0.5% to about 99% or more by weightof the composition, while liquid formulations may generally comprisefrom about 0.5% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10¹² cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

2.12 Chemical and Pharmaceutical Compositions

The inventors contemplate in addition to treating plants and theirenvironments with the insecticidal compositions of the invention, thetreatment of animals and their surroundings will also be possible withcertain chimeric crystal proteins which have activity against insectswhich infest animals and their environment.

The inventors particularly contemplate the use of chimeric crystalproteins in the formulation of bioinsecticides comprising specificchimeric δ-endotoxin proteins which have insecticidal activity againstone or more insects which infest pets, livestock, and other domesticanimals. The formulation of pharmaceutical compositions which may begiven to animals as prophylaxis andlor treatment of infestation by suchinsects, and in particular mosquitoes, flies, fleas, and related insectswill be particularly useful in treating such infestations. Insects asdescribed in detail in U.S. Pat. No. 5,449,681, incorporated herein byreference, may be particularly susceptible to treatment in this manner.Such insects include members of the Genera Culex, Culiseta, Bovicola,Callitroga, Chrysops, Cimes, Ctenocephalis, Ctenocephaledes,Dermatophilus, Dermatobia, and Damalinia among others.

Means for administering insecticidal compositions to an animal arewell-known in the art. U.S. Pat. No. 5,416,102 (specificallyincorporated herein by reference) provides excellent teaching formethods and formulations for preventing insect infestation using aninsecticidal composition. Veterinary-approved formulations may bedelivered in a variety of methods depending upon the particularapplication.

It is further contemplated that in addition to topical administration ofthe pharmaceutical compositions disclosed, systemic administration mayin some cases be preferable or desirable. For oral administration, thecompositions may be formulated with an inert diluent or with anassimilatable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltables, troches, capsules, elixirs, suspensions, syrups, wafers, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of the unit. The amount of active compounds insuch therapeutically useful compositions is such that a suitable dosagewill be obtained.

For oral prophylaxis, the crystal protein may be incorporated withexcipients and used in the form of a gel, paste, powder, pill, tablet,capsule, or slurry which may be given to the animal for ingestion.Alternatively the compositions may be formulated as an additive to petfoods, treats, or other edible formulations. When formulated as a tabletor capsule, or the like, the composition may also contain the following:a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients,such as dicalcium phosphate; a disintegrating agent, such as cornstarch, potato starch, alginic acid and the like; a lubricant, such asmagnesium stearate; and a sweetening agent, such as sucrose, lactose orsaccharin may be added or a flavoring agent to make the composition morepallatable to the animal being treated. One such means for deliveringprophylactics to an animal is a sauce as described in U.S. Pat. No.4,702,914 (specifically incorporated herein by reference).

When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compounds maybe incorporated into sustained-release preparation and formulations.

Alternatively, the pharmaceutical compositions disclosed herein may beadministered parenterally, intramuscularly, or even intraperitoneally.Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, and/orvegetable oils. Proper fluidity may be maintained, for example, by theuse of a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial ad antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

When systemic administration is desired, e.g., parenteral administrationin an aqueous solution, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition, size, and type of animal being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas creams, lotions, sprays, dips, emulsions, colloids, or alternatively,when systemic administration is desirable, injectable solutions, drugrelease capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a animal. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The inventors further contemplate the use of chimeric crystal proteinsof the present invention as active ingredients in pharmaceuticalcompositions for administration to the living areas and environment ofan animal to prevent, lessen, or reduce the infestation of susceptibleinsects in such an area. The chimeric proteins or a suspension of cellswhich express the particular chimeric protein(s) may be formulated in apowder, spray, fog, granule, rinse, shampoo, dip, etc. suitable foradministration to the living quarters, bedding materials, or environmentof the animal using techniques which are known to those of skill in theart of veterinary insecticide formulations. An example of oralformulation of veterinary insecticides is found in the teachings of U.S.Pat. No. 5,416,102.

2.13 Antibody Compositions and Methods for Producing

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the crystalproteins disclosed herein. Means for preparing and characterizingantibodies are well known in the art (See, e.g., Harlow and Lane, 1988;incorporated herein by reference). The methods for generating monoclonalantibodies (mAbs) generally begin along the same lines as those forpreparing polyclonal antibodies. Briefly, a polyclonal antibody isprepared by immunizing an animal with an immunogenic composition inaccordance with the present invention and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits; a rabbitis a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal. The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques suchas those exemplified in U.S. Pat. No. 4,196,265 (specificallyincorporated herein by reference). Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified crystal protein, polypeptide orpeptide. The immunizing composition is administered in a mannereffective to stimulate antibody producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep frogcells is also possible. The use of rats may provide certain advantages(Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. The wild-type δ-endotoxins and the relevant restriction sitesthat were used to construct the hybrid δ-endotoxins pertinent to theinvention are diagrammed in FIG. 1A. Only the DNA encoding theδ-endotoxin that is contained on the indicated plasmid (identified bythe “pEG” prefix) is shown. The B. thuringiensis strains containing theindicated plasmids are identified by the “EG” prefix. The hybridδ-endotoxins described in the invention are diagrammed in FIG. 1B andare aligned with the wild-type δ-endotoxins in FIG. 1A.

FIG. 2. An equal amount of each washed sporulated B. thuringiensisculture was analyzed by SDS-PAGE. Lane a: control Cry1Ac producing B.thuringiensis strain EG11070, b: EG11060, c: EG11062, d: EG11063, e:EG11065, f: EG11067, g: EG11071, h: EG11073, i: EG11074, j: EG11088, k:EG11090, and 1: EG11091.

FIG. 3. Solubilized hybrid δ-endotoxins were exposed to trypsin for 0,15, 30, 60, and 120 minutes. The resulting material was analyzed bySDS-PAGE. The amount of active δ-endotoxin fragment remaining wasquantitated by scanning densitometry using a Molecular Dynamics model300A densitometer. The percent active toxin remaining was plotted versustime. Wild-type Cry1Ac δ-endotoxin (open box) served as the control.

4. BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is oligonucleotide primer A

SEQ ID NO:2 is oligonucleotide primer B.

SEQ ID NO:3 is oligonucleotide primer C

SEQ ID NO:4 is oligonucleotide primer D.

SEQ ID NO:5 is oligonucleotide primer E.

SEQ ID NO:6 is oligonucleotide primer F.

SEQ ID NO:7 is oligonucleotide primer G.

SEQ ID NO:8 is oligonucleotide primer H.

SEQ ID NO:9 is the nucleotide and deduced amino acid sequences of theEG11063 hybrid δ-endotoxin.

SEQ ID NO:10 denotes the three-letter abbreviation form of the aminoacid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide and deduced amino acid sequences of theEG11074 hybrid δ-endotoxin.

SEQ ID NO:12 denotes the three-letter abbreviation form of the aminoacid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:11.

SEQ ID NO:13 is the nucleotide and deduced amino acid sequences of theEG11735 hybrid δ-endotoxin.

SEQ ID NO:14 denotes the three-letter abbreviation form of the aminoacid sequence for the hybrid δ-endotoxin specified in SEQ ID NO:13.

SEQ ID NO:15 is the 5′ exchange site for pEG1065, pEG1070, and pEG1074.

SEQ ID NO:16 is the 5′ exchange site for pEG1067, pEG1072, and pEG1076.

SEQ ID NO:17 is the 5′ exchange site for pEG1068, pEG1077, and pEG365.

SEQ ID NO:18 is the 5′ exchange site for pEG1088 and pEG1092.

SEQ ID NO:19 is the 5′ exchange site for pEG1089 and the 3′ exchangesite for pEG1070 and pEG1072.

SEQ ID NO:20 is the 5′ exchange site for pEG1091.

SEQ ID NO:21 is the 3′ exchange site for pEG1065, pEG1067, pEG1068, andpEG1093, pEG365, and pEG378.

SEQ ID NO:22 is the 3′ exchange site for pEG1088.

SEQ ID NO:23 is oligonucleotide Primer I.

SEQ ID NO:24 is oligonucleotide Primer J.

SEQ ID NO:25 is the nucleic acid sequence and deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11092.

SEQ ID NO:26 is the three-letter abbreviation form of the amino acidsequence of the hybrid crystal protein produced by strain EG11092encoded by SEQ ID NO:25.

SEQ ID NO:27 is the nucleic acid sequence and the deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11751.

SEQ ID NO:28 is the three-letter abbreviation form of the amino acidsequence of the hybrid crystal protein produced by strain EG11751encoded by SEQ ID NO:27.

SEQ ID NO:29 is the nucleic acid sequence and the deduced amino acidsequence of the hybrid crystal protein-encoding gene of EG11091.

SEQ ID NO:30 is the three-letter abbreviation form of the amino acidsequence of the hybrid crystal protein produced by strain EG11091encoded by SEQ ID NO:29.

5. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

5.1 Methods for Culturing B. thuringiensis to Produce the Novel Proteins

The B. thuringiensis strains described herein may be cultured usingstandard known media and fermentation techniques. Upon completion of thefermentation cycle, the bacteria may be harvested by first separatingthe B. thuringiensis spores and crystals from the fermentation broth bymeans well known in the art. The recovered B. thuringiensis spores andcrystals can be formulated into a wettable powder, a liquid concentrate,granules or other formulations by the addition of surfactants,dispersants, inert carriers and other components to facilitate handlingand application for particular target pests. The formulation andapplication procedures are all well known in the art and are used withcommercial strains of B. thuringiensis (HD-1) active againstLepidoptera, e.g., caterpillars.

5.2 Recombinant Host Cells For Expression of the Novel Chimeric Genes

The nucleotide sequences of the subject invention can be introduced intoa wide variety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the sites of lepidopteran insects where they willproliferate and be ingested by the insects. The results is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B. thuringiensis toxin.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility or toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and Gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B. thuringiensisgene into the host, availability of expression systems, efficiency ofexpression, stability of the pesticide in the host, and the presence ofauxiliary genetic capabilities. Characteristics of interest for use as apesticide microcapsule include protective qualities for the pesticide,such as thick cell walls, pigmentation, and intracellular packaging orformation of inclusion bodies; leaf affinity; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, Streptomyces lividans and the like.

Treatment of the microbial cell, e.g., a microbe containing the B.thuringiensis toxin gene, can be by chemical or physical means, or by acombination of chemical and/or physical means, so long as the techniquedoes not deleteriously affect the properties of the toxin, nor diminishthe cellular capability in protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehye; anti-infectives, such as zephiran chloride andcetylpyridinium chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Lugol's iodine, Bouin's fixative,and Helly's fixatives, (see e.g., Humason, 1967); or a combination ofphysical (heat) and chemical agents that preserve and prolong theactivity of the toxin produced in the cell when the cell is administeredto a suitable host. Examples of physical means are short wavelengthradiation such as γ-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. The cells employed will usually be intactand be substantially in the proliferative form when treated, rather thanin a spore form, although in some instances spores may be employed.

Where the B. thuringiensis toxin gene is introduced via a suitablevector into a microbial host, and said host is applied to theenvironment in a living state, it is essential that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment (crop and other insect habitats) with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus,Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus, andAzotobacter vinlandii; and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans.

5.3 Definitions

The following words and phrases have the meanings set forth below.

Broad-Spectrum: refers to a wide range of insect species.

Broad-Spectrum Insecticidal Activity: toxicity towards a wide range ofinsect species.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Insecticidal Activity: toxicity towards insects.

Insecticidal Specificity: the toxicity exhibited by a crystal proteintowards multiple insect species.

Intraorder Specificity: the toxicity of a particular crystal proteintowards insect species within an Order of insects (e.g., OrderLepidoptera).

Interorder Specificity: the toxicity of a particular crystal proteintowards insect species of different Orders (e.g., Orders Lepidoptera andDiptera).

LC₅₀: the lethal concentration of crystal protein that causes 50%mortality of the insects treated.

LC₉₅: the lethal concentration of crystal protein that causes 95%mortality of the insects treated.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast or explant).

Structural Gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed Cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgene: An exogenous gene which when introduced into the genome of ahost cell through a process such as transformation, electroporation,particle bombardment, and the like, is expressed by the host cell andintegrated into the cells genome such that the trait or traits producedby the expression of the transgene is inherited by the progeny of thetransformed cell.

Transgenic Cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell. Exemplary transgenic cells includeplant calli derived from a transformed plant cell and particular cellssuch as leaf, root, stem, e.g., somatic cells, or reproductive (germ)cells obtained from a transgenic plant.

Transgenic Plant: A plant or progeny thereof derived from a transformedplant cell or protoplast, wherein the plant DNA contains an introducedexogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callous obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

5.4 Probes And Primers

In another aspect, DNA sequence information provided by the inventionallows for the preparation of relatively short DNA (or RNA) sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein gene sequence, e.g., asequence such as that shown in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. The ability of such nucleicacid probes to specifically hybridize to a crystal protein-encoding genesequence lends them particular utility in a variety of embodiments. Mostimportantly, the probes may be used in a variety of assays for detectingthe presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCR™ using such primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes sequences that are complementary to at leasta 14 to 30 or so long nucleotide stretch of a crystal protein-encodingsequence, such as that shown in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. A size of at least 14nucleotides in length helps to ensure that the fragment will be ofsufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 14 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. Nos. 4,683,195, and 4,683,202, each specifically incorporatedherein by reference, or by excising selected DNA fragments fromrecombinant plasmids containing appropriate inserts and suitablerestriction sites.

5.5 Expression Vectors

The present invention contemplates an expression vector comprising apolynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to an coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region.

As used herein, the term “operatively linked” means that a promoter isconnected to an coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region are well known inthe art.

Promoters that function in bacteria are well known in the art. Anexemplary and preferred promoter for the Bacillus crystal proteinsinclude the sigA, sigE, and sigK gene promoters. Alternatively, thenative, mutagenized, or recombinant crystal protein-encoding genepromoters themselves can be used.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in plants. Promoters that function in plants are also wellknown in the art. Useful in expressing the polypeptide in plants arepromoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

A promoter is also selected for its ability to direct the transformedplant cell's or transgenic plant's transcriptional activity to thecoding region. Structural genes can be driven by a variety of promotersin plant tissues. Promoters can be near-constitutive, such as the CaMV35S promoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S,increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callous, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The Lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983; Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yanget al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), cornlight harvesting complex (Simpson, 1986), corn heat shock protein (Odellet al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986;Cashmore et al., 1983), Ti plasmidmannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) andPotato patatin (Wenzler et al., 1989). Preferred promoters are thecauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunitRuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). Plasmid pCaMVCN (available fromPharnacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV35S promoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, the disclosures of which are eachspecifically incorporated herein by reference. Those vectors can bemodified to include a coding sequence in accordance with the presentinvention.

A variety of methods has been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a chimeric B.thuringiensis crystal protein-encoding gene. In preferred embodiments,such a polypeptide has the amino acid residue sequence of SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30;or a functional equivalent of one or more of those sequences. Inaccordance with such embodiments, a coding region comprising the DNAsequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQID NO:27, or SEQ ID NO:29 is also preferred.

5.6 Transformed or Transgenic Plant Cells

A bacterium, a yeast cell, or a plant cell or a plant transformed withan expression vector of the present invention is also contemplated. Atransgenic bacterium, yeast cell, plant cell or plant derived from sucha transformed or transgenic cell is also contemplated. Means fortransforming bacteria and yeast cells are well known in the art.Typically, means of transformation are similar to those well known meansused to transform other bacteria or yeast such as E. coli orSaccharomyces cerevisiae.

Methods for DNA transformation of plant cells includeAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos and particle bombardment. Each of these methodshas distinct advantages and disadvantages. Thus, one particular methodof introducing genes into a particular plant strain may not necessarilybe the most effective for another plant strain, but it is well knownwhich methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973);(2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viralvectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a;1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;Wagner et al., 1992).

5.6.1 Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of clones genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callous,or alternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

5.6.2 Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

The methods of particle-mediated transformation is well-known to thoseof skill in the art. U.S. Pat. No. 5,015,580 (specifically incorporatedherein by reference) describes the transformation of soybeans using sucha technique.

5.6.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). The genetic engineering of cottonplants using Agrobacterium-mediated transfer is described in U.S. Pat.No. 5,004,863 (specifically incorporated herein by reference), while thetransformation of lettuce plants is described in U.S. Pat. No. 5,349,124(specifically incorporated herein by reference). Further, theintegration of the Ti-DNA is a relatively precise process resulting infew rearrangements. The region of DNA to be transferred is defined bythe border sequences, and intervening DNA is usually inserted into theplant genome as described (Spielmann et al., 1986; Jorgensen et al.,1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, for example, Bytebier etal., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced carboxylase activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

5.7 Production of Insect-Resistant Transgenic Plants

Thus, the amount of a gene coding for a polypeptide of interest (i.e., abacterial crystal protein or polypeptide having insecticidal activityagainst one or more insect species) can be increased in plant such ascorn by transforming those plants using particle bombardment methods(Maddock et al., 1991). By way of example, an expression vectorcontaining a coding region for a B. thuringiensis crystal protein and anappropriate selectable marker is transformed into a suspension ofembryonic maize (corn) cells using a particle gun to deliver the DNAcoated on microprojectiles. Transgenic plants are regenerated fromtransformed embryonic calli that express the disclosed insecticidalcrystal proteins. Particle bombardment has been used to successfullytransform wheat (Vasil et al., 1992).

DNA can also be introduced into plants by direct DNA transfer intopollen as described (Zhou et al., 1983; Hess, 1987; Luo et al., 1988).Expression of polypeptide coding genes can be obtained by injection ofthe DNA into reproductive organs of a plant as described (Pena et al.,1987). DNA can also be injected directly into the cells of immatureembryos and the rehydration of desiccated embryos as described (Neuhauset al., 1987; Benbrook et al., 1986).

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 details the creation of genetically transformed lettucecells and plants resulting therefrom which express hybrid crystalproteins conferring insecticidal activity against Lepidopteran larvae tosuch plants.

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

A transgenic plant of this invention thus has an increased amount of acoding region (e.g., a cry gene) that encodes the Cry polypeptide ofinterest. A preferred transgenic plant is an independent segregant andcan transmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for, by way of example, increased insecticidal capacityagainst Coleopteran insects, preferably in the field, under a range ofenvironmental conditions. The inventors contemplate that the presentinvention will find particular utility in the creation of transgeniccorn, wheat, oats, barley, other grains, vegetables, fruits, fruittrees, berries, turf grass, ornamentals, shrubs and trees.

6. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

6.1 Example 1 Construction of Hybrid B. thuringiensis δ-Endotoxins

The B. thuringiensis shuttle vectors pEG853, pEG854, and pEG857 whichare used in the present invention are described by Baum et al., 1990.The plasmid pEG857 contains the Cry1Ac gene cloned into pEG853 as anSph1-BamHI DNA fragment. pEG1064 was constructed for purposes of thepresent invention in such a way that the KpnI site within the Cry1Acgene was preserved and the KpnI site in the pEG857 multiple cloning site(MCS) was eliminated. This was accomplished by sequentially subjectingpEG857 DNA to limited KpnI digestion so that only one KpnI site is cut,filling in the KpnI 5′ overhang by Klenow fragment of DNA polymerase Ito create blunt DNA ends, and joining the blunt ends of DNA by T4 DNAligase. pEG318 contains the Cry1F gene (Chambers et al., 1991) clonedinto the XhoI site of pEG854 as a XhoI-SalI DNA fragment. pEG315contains the Cry1C gene from strain EG6346 (Chambers et al., 1991)cloned into the XhoI-BamHI sites of pEG854 as a SalI-BamHI DNA fragment.

FIG. 1A shows a schematic representation of the DNA encoding thecomplete Cry1Ac, Cry1Ab, Cry1C, and Cry1F genes contained onpEG854/pEG1064, pEG20, pEG315, and pEG318, respectively. Uniquerestriction sites that were used in constructing certain hybrid genesare also shown. FIG. 1B shows a schematic representation of hybrid genespertaining to the present invention. In some cases standard PCR™amplification with mutagenic oligonucleotide primers were used toincorporate appropriate restrictions sites into DNA fragments used forhybrid gene construction. Certain hybrid gene constructions could not beaccomplished by restriction fragment subcloning. In those instances,PCR™ overlap extension (POE) was used to construct the desired hybridgene (Horton et al., 1989). The following oligonucleotide primers(purchased from Integrated DNA Technologies Inc., Coralville, Iwoa) wereused:

Primer A

5′-GGATAGCACTCATCAAAGGTACC-3′ (SEQ ID NO:1)

Primer B

5′-GAAGATATCCAATTCGAACAGTTTCCC-3′ (SEQ ID NO:2)

Primer C

5′-CATATTCTGCCTCGAGTGTTGCAGTAAC-3′ (SEQ ID NO:3)

Primer D

5′-CCCGATCGGCCGCATGC-3′ (SEQ ID NO:4)

Primer E

5′-CATTGGAGCTCTCCATG-3′ (SEQ ID NO:5)

Primer F

5′-GCACTACGATGTATCC-3′ (SEQ ID NO:6)

Primer G

5′-CATCGTAGTGCAACTCTTAC-3′ (SEQ ID NO:7)

Primer H

5′-CCAAGAAAATACTAGAGCTCTTGTTAAAAAAGGTGTTCC-3′ (SEQ ID NO:8)

Primer I

5′-ATTTGAGTAATACTATCC-3′ (SEQ ID NO:23)

Primer J

5′-ATTACTCAAATACCATTGG-3′ (SEQ ID NO:24)

The plasmids described in FIG. 1B containing the hybrid δ-endotoxingenes pertinent to this invention are described below. Isolation orpurification of DNA fragments generated by restriction of plasmid DNA,PCR™ amplification, or POE refers to the sequential application ofagarose-TAE gel electrophoresis and use of the Geneclean Kit (Bio 101)following the manufacturer's recommendation. pEG1065 was constructed byPCR™ amplification of the Cry1F DNA fragment using primer pair A and Band pEG318 as the DNA template. The resulting PCR™ product was isolated,cut with AsulI and KpnI, and used to replace the correspondingAsuII-KpnI DNA fragment in pEG857. Plasmid pEG1067 was constructed usingPOE and DNA fragments SauI-KpnI of Cry1F and AsuII-ClaI of Cry1Ac thatwere isolated from pEG318 and pEG857, respectively. The resulting POEproduct was PCR™ amplified with primer pair A and B, cut with AsuII andKpnI, and used to replace the corresponding AsuII-KpnI fragment inpEG857.

pEG1068 was constructed by replacing the SacI-KpnI DNA fragment ofCry1Ac isolated from pEG857 with the corresponding SacI-KpnI DNAfragment isolated from Cry1F (pEG318). pEG1070 was constructed byreplacing the SacI-KpnI DNA fragment isolated from pEG1065 with thecorresponding SacI-KpnI DNA fragment isolated from Cry1Ac (pEG857).pEG1072 was constructed by replacing the SacI-Kpnl DNA fragment isolatedfrom pEG1067 with the corresponding SacI-KpnI DNA fragment isolated fromCry1Ac (pEG857). pEG1074, pEG1076, and pEG1077 were constructed byreplacing the SphI-XhoI DNA fragment from pEG1064 with the PCR™amplified SphI-XhoI DNA fragment from pEG1065, pEG1067, pEG1068,respectively, using primer pairs C and D. pEG1089 was constructed byreplacing the SphI-SacI DNA fragment of pEG1064 with the isolated andSphI and SacI cut PCR™ product of Cry1F that was generated using primerpair D and E and the template pEG318.

pEG1091 was constructed by replacing the SphI-SacI DNA fragment ofpEG1064 with the isolated and SphI and SacI cut PCR™ product of Cry1Cthat was generated using primer pair D and H and the template pEG315.

pEG1088 was constructed by POE using a Cry1Ac DNA fragment generatedusing primer pair B and F and a Cry1C DNA fragment generated usingprimer pair A and G. The SacI-KpnI fragment was isolated from theresulting POE product and used to replace the corresponding SacI-KpnIfragment in pEG1064.

pEG365 was constructed by first replacing the SphI-KpnI DNA fragmentfrom pEG1065 with the corresponding Cry1Ab DNA fragment isolated frompEG20 to give pEG364. The SacI-KpnI DNA fragment from pEG364 was thenreplaced with the corresponding Cry1F DNA fragment isolated from pEG318.

pEG1092 was constructed by replacing the KpnI-BamHI DNA fragment frompEG1088 with the corresponding DNA fragment isolated from pEG315.pEG1092 is distinct from the cry1Ab/cry1C hybrid δ-endotoxin genedisclosed in Intl. Pat. Appl. Publ. No. WO 95/06730.

pEG1093 was constructed by replacing the SphI-AsuII DNA fragment frompEG1068 with the corresponding SphI-AsuII DNA fragment isolated frompEG20.

pEG378 was constructed by POE using a cry1Ac DNA fragment generatedusing primer pair B and I using pEG857 as the template and a cry1F DNAfragment generated using primer pair A and J using pEG318 as thetemplate. The resulting POE product was cut with AsuII and KpnI and theresulting isolated DNA fragment used to replace the correspondingAsuII-KpnI DNA fragment in pEG1064.

6.2 Example 2 Production of the Hybrid Toxins in B. thuringiensis

The plasmids encoding the hybrid toxins described in Example 1 weretransformed into B. thuringiensis as described by Mettus and Macaluso,1990. The resulting B. thuringiensis strains were grown in 50 ml of C-2medium until the culture was fully sporulated and lysed (approximately48 hr.). Since crystal formation is a prerequisite for efficientcommercial production of δ-endotoxins in B. thuringiensis, microscopicanalysis was used to identify crystals in the sporulated cultures (TABLE3).

TABLE 3 Crystal Formation by the Hybrid δ-Endotoxins NRRL^(a) NRRLCrystal Accession Deposit Parent δ- For- Strain Number Date PlasmidEndotoxins mation EG11060 pEG1065 Cry1Ac + Cry1F + EG11062 pEG1067Cry1Ac + Cry1F + EG11063 B-21579 May 24, pEG1068 Cry1Ac + Cry1F +EG11065 1996 pEG1070 Cry1Ac + Cry1F − EG11067 pEG1072 Cry1Ac + Cry1F −EG11071 pEG1074 Cry1Ac + Cry1F + EG11073 pEG1076 Cry1Ac + Cry1F +EG11074 B-21580 May 24, pEG1077 Cry1Ac + Cry1F + 1996 Cry1F EG11087pEG1088 Cry1Ac + Cry1C − EG11088 pEG1089 Cry1F + Cry1Ac − EG11090pEG1091 Cry1C + Cry1Ac − EG11091 pEG1092 Cry1Ac + Cry1C + EG11092B-21635 Oct. 21, pEG1093 Cry1Ab + + 1996 Cry1Ac + Cry1F EG11735 B-21581May 24, pEG365 Cry1Ab + + 1996 Cry1F + Cry1Ac EG11751 B-21636 Oct. 21,pEG378 Cry1Ac + Cry1F + 1996 ^(a)The subject cultures have beendeposited under conditions that assure that access to the cultures willbe available during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits areavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny, are filed.However, it should be #understood that the availability of a depositdoes not constitute a license to practice the subject invention inderogation of patent rights granted by governmental action. The subjectculture deposits will be stored and made available to the public inaccord with the provisions of the Budapest Treaty for the Deposit ofMicroorganisms, i.e., they will be stored with all the care necessary tokeep them viable and uncontaminated for a period of at least five yearsafter the #most recent request for the finishing of a sample of thedeposit, and in any case, for a period of at least 30 (thirty) yearsafter the date of deposit or for the enforceable life of any patentwhich may issue disclosing the cultures. The depositor acknowledges theduty to replace the deposits should the depository be unable to furnisha sample when requested, due to the condition of the deposits. Allrestrictions on the availability to the public of the subject culturedeposits #will be irrevocably removed upon the granting of a patentdisclosing them. Cultures shown in Table 3 were deposited in thepermanent collection of the Agricultural Research Service CultureCollection, Northern Regional Research Laboratory (NRRL), located at1815 N. University Street, Peoria, IL 61604, under the terms of theBudapest Treaty.--

The δ-endotoxin production for some of the B. thuringiensis strainsspecified in TABLE 3 was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described byBaum et al., 1990. Equal volume cultures of each B. thuringiensis strainwere grown in C-2 medium until fully sporulated and lysed. The cultureswere centrifuged and the spore/crystal pellet was washed twice withequal volumes of distilled deionized water. The final pellet wassuspended in half the culture volume of 0.005% Triton X-100®. An equalvolume of each washed culture was analyzed by SDS-PAGE as shown in FIG.2.

The majority of hybrids involving Cry1Ac and Cry1F formed stablecrystals in B. thuringiensis A notable exception is EG11088 in which theactive toxin fragment would be the reciprocal exchange of EG1 1063. Twoof the three hybrids involving Cry1Ac and Cry1C, EG11087 and EG11090,failed to produce crystal in B. thuringiensis even though thesereciprocal hybrids mimic the activated toxin fragments ofcrystal-forming EG11063 and EG11074.

Every strain that was examined by SDS-PAGE produced some level ofδ-endotoxin. As expected, however, those cultures identified as crystalnegative produced very little protein (e.g., lane e: EG11065, lane f:EG11067, lane j: EG11088, and lane k: EG11090). For reference, typicalyields from a crystal forming δ-endotoxin is shown for Cry1Ac (lane a).Several hybrid δ-endotoxins produce comparable levels of proteinincluding EG11060 (lane b), EG11062 (lane c), EG11063 (lane d; SEQ IDNO:10), and EG11074 (lane i; SEQ ID NO:12). The data clearly show thatefficient hybrid δ-endotoxin production in B. thuringiensis isunpredictable and varies depending on the parent δ-endotoxins used toconstruct the hybrid.

6.3 Example 3 Proteolytic Processing of the Hybrid δ-Endotoxins

Proteolytic degradation of the protoxin form of the δ-endotoxin to astable active toxin occurs once δ-endotoxin crystals are solubilized inthe larval midgut. One measure of the potential activity of δ-endotoxinsis the stability of the active δ-endotoxin in a proteolytic environment.To test the proteolytic sensitivity of the hybrid δ-endotoxins,solubilized toxin was subjected to trypsin digestion. The δ-endotoxinswere purified from sporulated B. thuringiensis cultures and quantifiedas described by Chambers et al., 1991. Exactly 250 μg of each hybridδ-endotoxin crystal was solubilized in 30 mM NaHCO₃, 10 mM DTT (totalvolume 0.5 ml). Trypsin was added to the solubilized toxin at a 1:10ratio. At appropriate time points 50 μl aliquots were removed to 50 μlLaemmli buffer, heated to 100° C. for 3 min., and frozen in a dry-iceethanol bath for subsequent analysis. The trypsin digests of thesolubilized toxins were analyzed by SDS-PAGE and the amount of activeδ-endotoxin at each time point was quantified by densitometry. A graphicrepresentation of the results from these studies are shown in FIG. 3.

The wild-type Cry1Ac is rapidly processed to the active δ-endotoxinfragment that is stable for the duration of the study. The hybridδ-endotoxins from EG11063 and EG11074 are also processed to activeδ-endotoxin fragments which are stable for the duration of the study.The processing of the EG11063 δ-endotoxin occurs at a slower rate and ahigher percentage of this active δ-endotoxin fragment remains at eachtime point. Although the hybrid δ-endotoxins from EG11060 and EG11062are process to active δ-endotoxin fragments, these fragments are moresusceptible to further cleavage and degrade at various rates during thecourse of the study. The 5′ exchange points between Cry1Ac and Cry1F forthe EG11062 and EG11063 δ-endotoxins result in toxins that differ byonly 21 amino acid residues (see FIG. 1). However, the importance ofmaintaining Cry1Ac sequences at these positions is evident by the morerapid degradation of the EG11062 δ-endotoxin. These data demonstratethat different hybrid δ-endotoxins constructed using the same parentalδ-endotoxins can vary significantly in biochemical characteristics suchas proteolytic stability.

6.4 Example 4 Bioactivity of the Hybrid δ-Endotoxins

B. thuringiensis cultures expressing the desired δ-endotoxin were grownuntil fully sporulated and lysed and washed as described in Example 2.The δ-endotoxin levels for each culture were quantified by SDS-PAGE asdescribed by Baum et al., 1990. In the case of bioassay screens, asingle appropriate concentration of each washed δ-endotoxin culture wastopically applied to 32 wells containing 1.0 ml artificial diet per well(surface area of 175 mm²) A single neonate larvae was placed in each ofthe treated wells and the tray covered by a clear perforated mylarsheet. Larvae mortality was scored after 7 days of feeding and percentmortality expressed as the ratio of the number of dead larvae to thetotal number of larvae treated (32).

In the case of LC₅₀ determinations (δ-endotoxin concentration giving 50%mortality), δ-endotoxins were purified from the B. thuringiensiscultures and quantified as described by Chambers et al., 1991. Eightconcentrations of the δ-endotoxins were prepared by serial dilution in0.005% Triton X-100® and each concentration was topically applied towells containing 1.0 ml of artificial diet. Larvae mortality was scoredafter 7 days of feeding (32 larvae for each δ-endotoxin concentration).In all cases the diluent served as the control.

A comparison of the Cry1A/Cry1F hybrid toxins by bioassay screens isshown in TABLE 4. The hybrid δ-endotoxins from strains EG11063 andEG11074 maintain the activities of the parental Cry1Ac and Cry1Fδ-endotoxins. Furthermore, the hybrid δ-endotoxin from EG11735 maintainsthe activity of its parental Cry1Ab and Cry1F δ-endotoxins. Theδ-endotoxins produce by strains EG11061, EG11062, EG11071, and EG11073have no insecticidal activity on the insect larvae tested despite 1)being comprised of at least one parental δ-endotoxin that is activeagainst the indicated larvae and 2) forming stable, well-definedcrystals in B. thuringiensis. These results demonstrate theunpredictable nature of hybrid toxin constructions.

For the data in TABLE 4. All strains were tested as washed sporulatedcultures. For each insect tested, equivalent amounts of δ-endotoxinswere used and insecticidal activity was based on the strain showing thehighest percent mortality (++++).

TABLE 4 Bioassay Screens of Hybrid Cry1A/Cry1F δ -Endotoxins H. StrainS. frugiperda S. exigua H. virescens zea O. nubilalis Cry1Ac − − ++++++++ +++ Cry1F ++++ ++ ++ ++ ++ Cry1Ab ++ + +++ ++ +++ EG11060 − − − − −EG11062 − − − − − EG11063 ++++ ++++ +++ +++ ++++ EG11071 − − − − −EG11073 − − − − − EG11074 ++++ ++++ +++ +++ ++++ EG11090 − +++ − − −EG11091 ++++ ++++ − − N.D.^(a) EG11092 ++++ ++++ +++ +++ N.D.^(a)EG11735 ++++ ++++ +++ +++ N.D.^(a) EG11751 N.D.^(a) ++++ N.D.^(a) ++++N.D.^(a) ^(a)N.D. = not determined.

The δ-endotoxins described in FIG. 1 and that demonstrated insecticidalactivity in bioassay screens were tested as purified crystals todetermine their LC₅₀ (see TABLE 5). The δ-endotoxins purified fromstrains EG11063, EG11074, EG11091, and EG11735 all show increased armyworm (S. frugiperda and S. exigua) activity compared to any of thewild-type δ-endotoxins tested. The EG11063 and EG11074 δ-endotoxinswould yield identical active toxin fragments (refer to FIG. 1B) which isevident by their similar LC50 values on the insects examined. Anunexpected result evident from these data is that a hybrid δ-endotoxinsuch as EG11063, EG11092, EG11074, EG11735, or EG11751 can retain theactivity of their respective parental δ-endotoxins, and, against certaininsects such as S. exigua, can have activity far better than eitherparental δ-endotoxin. This broad range of insecticidal activity at dosesclose to or lower than the parental δ-endotoxins, along with thewild-type level of toxin production (see Example 2), make these proteinsparticularly suitable for production in B. thuringiensis. Although theEG11091 derived δ-endotoxin has better activity against S. frugiperdaand S. exigua than its parental δ-endotoxins, it has lost the H.virescens and H. zea activity attributable to its Cry1Ac parent. Thisrestricted host range along with lower toxin yield observed for theEG11091 δ-endotoxin (see Example 2) make it less amenable to productionin B. thuringiensis

TABLE 5 LC⁵⁰ Values for the Purified Hybrid δ-Endotoxin^(a) S. H. O.Toxin S. frugiperda exigua virescens H. zea nubilalisCry1Ac >10000 >10000 9 100 23 Cry1Ab 1435 4740 118 400 17 Cry1C >10000490 >10000 >10000 >10000 Cry1F 1027 3233 54 800 51 EG11063 550 114 33 807 (Cry1Ac/1F) EG11074 468 77 25 76 9 (Cry1Ac/1F) EG11090 21 21219 >10000 nd (Cry1Ac/1C)

In TABLE 5, the LC₅₀ values are expressed in nanograms of purifiedδ-endotoxin per well (175 mm²) and are the composite values for 2 to 6replications. nd=not determined.

TABLE 6 DNA Exchange Sites for Cryl Hybrid δ-Endotoxins Plasmid SEQ IDNO: 5′ Exchange Site SEQ ID NO: 3′ Exchange Site pEG1065 15TATCCAATTCGAACGTCATC 21 ACTACCAGGTACCTTTGATG pEG1067 16TTTAGTCATCGATTAAATCA 21 ACTACCAGGTACCTTTGATG pEG1068 17ATAATAAGAGCTCCAATGTT 21 ACTACCAGGTACCTTTGATG pEG1070 15TATCCAATTCGAACGTCATC 19 TCATGGAGAGCTCCTATGTT pEG1072 16TTTAGTCATCGATTAAATCA 19 TCATGGAGAGCTCCTATGTT pBG1074 15TATCCAATTCGAACGTCATC TGCAACACTCGAGGCTGAAT pEG1076 16TTTAGTCATCGATTAAATCA TCCAACACTCGAGGCTGAAT pEG1077 17ATAATAAGAGCTCCAATGTT TGCAACACTCGAGGCTGAAT pEG1088 18TACATCGTAGTGCAACTCTT 22 ACTACCGGGTACCTTTGATA pEG1089 19TCATGGAGAGCTCCTATGTT — pEG1091 20 TTAACAAGAGCTCCTATGTT — pEG1092 18TACATCGTAGTGCAACTCTT — pEG1093 21 — 21 ACTACCAGGTACCTTTGATG pEG365 17ATAATAAGAGCTCCAATGTT 21 ACTACCAGGTACCTTTGATG pEG378 32 — 21ACTACCAGGTACCTTTGATG

TABLE 6 describes the DNA surrounding the 5′ and 3′ exchange points forthe hybrid δ-endotoxins which are pertinent to the present invention. Asevident by the SEQ ID NO, certain hybrid δ-endotoxins share exchangesites. In certain instances, the exchange site is indistinguishable fromone of the parent δ-endotoxins. This is the case for the 3′ exchangesite for pEG1074, pEG1076, and pEG1077 and therefore, they have not beenassigned a separate sequence identifier (SEQ ID NO.).

To examine the effect of other small changes in the exchange site chosenfor hybrid endotoxin construction, the activity of EG11751 and EG11063on S. exigua and H. zea were compared (TABLE 7). The data clearly showthat hybrid δ-endotoxin improvements can be made by altering theexchange site between the two parental δ-endotoxins. In this example,the exchange site in the EG11751 δ-endotoxin was moved 75 base pairs 3′compared to the EG11063 δ-endotoxin and results in improved insecticidalactivity. Although no significant improvement in S. exigua activity isobserved between EG11063 and EG11751, a significant improvement in H.zea activity of almost 4-fold is observed for EG11751. It is importantto note that improvements in hybrid δ-endotoxin bioactivity by alteringexchange sites is unpredictable. In the case of EG11062, moving theexchange site 63 base pairs 5′ of the EG11063 exchange site abolishesinsecticidal activity as shown in TABLE 5.

TABLE 7 Bioactivity of EG11063 and EG11751 LC₅₀ Values for WashedSporulated Cultures B.t. Strain S. exigua H. zea EG11063 106 38 EG1175190 10

6.5 EXAMPLE 5 Amino Acid Sequences of the Novel Crystal Proteins

6.5.1 AMINO ACID SEQUENCE OF THE EG11063 CRYSTAL PROTEIN (SEQ ID NO:10)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.5.2 AMINO ACID SEQUENCE OF THE EG11074CRYSTAL PROTEIN (SEQ ID NO:12)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrIleThrAsnThrIleAspProGluArgIleThrGlnIleProLeuVaILysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsnValThrAspTyrHisIleAspGlnValSerAsnLeuValThrTyrLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSerAsnPheLysAspIeAsnArgGlnProGluArgGlyTrpGlyGlySerThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.5.3 AMINO ACID SEQUENCE OF THE EG11735CRYSTAL PROTEIN (SEQ ID NO:14)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleIleLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluGlySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyGluTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuIleSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspIleGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.5.4 AMINO ACID SEQUENCE OF THE EG11092CRYSTAL PROTEIN (SEQ ID NO:26)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaIleMetPheSerTrpThrHisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnIleCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.5.5 AMINO ACID SEQUENCE OF THE EG11751CRYSTAL PROTEIN (SEQ ID NO:23)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrIleIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerIleValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuIleSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsnIleIleAlaSerAspSerIleThrGlnIleProLeuValLysAlaHisThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerGlyGlyProPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArgTyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsnLysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAspArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerIleAsnGlnIleGlyIleLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeuAspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaIleHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.5.6 AMINO ACID SEQUENCE OF THE 11091CRYSTAL PROTEIN (SEQ ID NO:30)MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrProIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuIleSerAlaValTyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArgSerAlaThrLeuThrAsnThrIleAspProGluArgIleAsnGlnIleProLeuValLysGlyPheArgValTrpGlyGlyThrSerValIleThrGlyProGlyPheThrGlyGlyAspIleLeuArgArgAsnThrPheGlyAspPheValSerLeuGlnValAsnIleAsnSerProIleThrGlnArgTyrArgLeuArgPheArgTyrAlaSerSerArgAspAlaArgValIleValLeuThrGlyAlaAlaSerThrGlyValGlyGlyGlnValSerValAsnMetIleLeuGlnLysThrMetGluIleGlyGluAsnLeuThrSerArgThrPheArgTyrThrAspPheSerAsnProPheSerPheArgAlaAsnProAspIleIleGlyIleSerGluGlnProLeuPheGlyAlaGlySerIleSerSerGlyGluLeuTyrIleAspLysIleGluIleIleLeuAlaAspAlaThrPheGluAlaGluSerAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerSerAsnGlnIleGlyLeuLysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheArgGlyIleAsnArgGlnProAspArgGlyTrpArgGlySerThrAspIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrValAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaTyrThrArgTyrGluLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyrAsnAlaLysHisGluIleValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheThrLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuLeuGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGlnLeuGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspArgLeuGlnValAspThrAsnIleAlaMetIleHisAlaAlaAspLysArgValHisArgIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArgIlePheThrAlaTyrSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuLeuCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnHisArgSerValLeuValIleProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAspAsnThrAspGluLeuLysPheSerAsnCysValGluGluGluValTyrProAsnAsnThrValThrCysAsnAsnTyrThrGlyThrGlnGluGluTyrGluGlyThrTyrThrSerArgAsnGlnGlyTyrAspGluAlaTyrGlyAsnAsnProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluSerAsnArgGlyTyrGlyAspTyrThrProLeuProAlaGlyTyrValThrLysAspLeuGluTyrPheProGluThrAspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSer ValGluLeuLeuLeuMetGluGlu

6.6 EXAMPLE 6 DNA Sequences Encoding the Novel Crystal Proteins

6.6.1 DNA SEQUENCE ENCODING THE EG11063 CRYSTAL PROTEIN (SEQ ID NO:9)ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGTAAC CCT GAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACCCCA ATC GAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTTCCC GGT GCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTTGGT CCC TCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AACCAA AGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGACTA AGC AAT CTT TAT CAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCAGAT CCT ACT AAT CCA GCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GACATG AAC AGT GCC CTT ACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TATCAA GTT CCT CTT TTA TCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCAGTT TTG AGA GAT GTT TCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCGACT ATC AAT AGT CGT 576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACAGAT TAT GCT GTA 624 CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCGGAT TCT AGA 672 GAT TGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTAACT GTA 720 TTA GAT ATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TATCCA 768 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA816 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATCTAT ACG GAT GCT CAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATGGCT TCT CCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGAACT ATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAGGGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATAGGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TATGGA ACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTAGAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAAGGA TTT AGT CAT CGA TTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGTAAT AGT AGT GTA AGT ATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGTAGT GCA ACC CCT ACA AAT 1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCATTG GTA AAA GCA CAT 1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCCGGG TTT ACG GGA 1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCTTAT ACT ATT 1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGAATA CGC 1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGTGAA 1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TAA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531 6.6.2 DNA SEQUENCEENCODING THE EG11074 CRYSTAL PROTEIN (SEQ ID NO:11) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACAAAT 1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA CTC GAG GCT GAA TAT AAT CTG GAA AGA GCG CAG AAG GCG GTG1872 AAT GCG CTG TTT ACG TCT ACA AAC CAA CTA GGG CTA AAA ACA AAT GTA1920 ACG GAT TAT CAT ATT GAT CAA GTG TCC AAT TTA GTT ACG TAT TTA TCG1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT2064 TTC AAA GAC ATT AAT AGG CAA CCA GAA CGT GGG TGG GGC GGA AGT ACA2112 GGG ATT ACC ATC CAA GGA GGG GAT GAC GTA TTT AAA GAA AAT TAC GTC2160 ACA CTA TCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531 6.6.3 DNA SEQUENCEENCODING THE EG11735 CRYSTAL PROTEIN (SEQ ID NO:13) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 GGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGA GGA GAA TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACAAAT 1392 ACA ATT GAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3604 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3531 6.6.4 DNA SEQUENCEENCODING THE EG11092 CRYSTAL PROTEIN (SEQ ID NO:25) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT TCT CTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCA ATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACAAAT 1392 ACA ATT CAT CCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT CCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA ACA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534 6.6.5 DNA SEQUENCEENCODING THE EG11751 CRYSTAL PROTEIN (SEQ ID NO:27) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCT GAA TTT AATAAT 1392 ATA ATT GCA TCG GAT AGT ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT1440 ACA CTT CAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA1488 GGA GAT ATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT1536 GTT AAT ATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC1584 TAT GCC TCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA1632 CGG ATT TTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA1680 TTA ACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA1728 TTC CCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT1776 TCA GGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT1824 GCA ACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG1872 AAT GCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG1920 ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA1968 GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA2016 CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC2064 TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG2112 GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC2160 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA2208 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA2256 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC2304 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG2352 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA2400 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT2928 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG2976 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT3024 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT3120 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GAC GAA3168 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AAT AAC ACG3216 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TAC GGA GGT GCG3264 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCT TCC GTA CCA GCT3312 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3360 GAG AAT CCT TGT GAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA3408 CCA GTT GGT TAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT3456 AAG GTA TGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC3504 AGC GTG GAA TTA CTC CTT ATG GAG GAA TAG 3534 6.6.6 DNA SEQUENCEENCODING THE EG11091 CRYSTAL PROTEIN (SEQ ID NO:29) ATG GAT AAC AAT CCGAAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAATTC GCT AGG AAC CAA GCC 288 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TATCAA ATT TAC GCA GAA 336 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCAGCA TTA AGA GAA 384 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTT TTATCA GTA 480 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTG AGA GAT GTTTCA 528 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACT ATC AAT AGT CGT576 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624CGC TGG TAC AAT ACG GGA TTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 GATTGG GTA AGG TAT AAT CAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 TTA GATATC GTT GCT CTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 ATT CGA ACAGTT TCC CAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 TTA GAA AAT TTTGAT GGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 CTA TAT GGA ACT ATG GGA AAT GCAGCT CCA CAA CAA CGT ATT GTT GCT 1056 CAA CTA GGT CAG GGC GTG TAT AGA ACATTA TCG TCC ACT TTA TAT AGA 1104 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCA AATTTG CCA TCC GCT GTA 1200 TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG GAT GAAATA CCG CCA CAG 1248 AAT AAC AAC GTG CCA CCT AGG CAA GGA TTT AGT CAT CGATTA AGC CAT 1296 GTT TCA ATG TTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGTATA ATA 1344 AGA GCT CCT ATG TTC TCT TGG ATA CAT CGT AGT GCA ACT CTT ACAAAT 1392 ACA ATT GAT CCA GAG AGA ATT AAT CAA ATA CCT TTA GTG AAA GGA TTT1440 AGA GTT TGG GGG GGC ACC TCT GTC ATT ACA GGA CCA GGA TTT ACA GGA1488 GGG GAT ATC CTT CGA AGA AAT ACC TTT GGT GAT TTT GTA TCT CTA CAA1536 GTC AAT ATT AAT TCA CCA ATT ACC CAA AGA TAC CGT TTA AGA TTT CGT1584 TAC GCT TCC AGT AGG GAT GCA CGA GTT ATA GTA TTA ACA GGA GCG GCA1632 TCC ACA GGA GTG GGA GGC CAA GTT AGT GTA AAT ATG CCT CTT CAG AAA1680 ACT ATG GAA ATA GGG GAG AAC TTA ACA TCT AGA ACA TTT AGA TAT ACC1728 GAT TTT AGT AAT CCT TTT TCA TTT AGA GCT AAT CCA GAT ATA ATT GGG1776 ATA AGT GAA CAA CCT CTA TTT GGT GCA GGT TCT ATT AGT AGC GGT GAA1824 CTT TAT ATA GAT AAA ATT GAA ATT ATT CTA GCA GAT GCA ACA TTT GAA1872 GCA GAA TCT GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCC CTG TTT1920 ACT TCT TCC AAT CAA ATC GGG TTA AAA ACC GAT GTG ACG GAT TAT CAT1968 ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA TTT TGT2016 CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA2064 CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC TTC AGA GGG ATC2112 AAT AGA CAA CCA GAC CGT GGC TGG AGA GGA AGT ACA GAT ATT ACC ATC2160 CAA GGA GGA GAT GAC GTA TTC AAA GAG AAT TAC GTC ACA CTA CCG GGT2208 ACC GTT GAT GAG TGC TAT CCA ACG TAT TTA TAT CAG AAA ATA GAT GAG2256 TCG AAA TTA AAA GCT TAT ACC CGT TAT GAA TTA AGA GGG TAT ATC GAA2304 GAT AGT CAA GAC TTA GAA ATC TAT TTG ATC CGT TAC AAT GCA AAA CAC2352 GAA ATA GTA AAT GTG CCA GGC ACG GGT TCC TTA TGG CCG CTT TCA GCC2400 CAA AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC2448 CTT GAA TGG AAT CCT GAT CTA GAT TGT TCC TGC AGA GAC GGG GAA AAA2496 TGT GCA CAT CAT TCC CAT CAT TTC ACC TTG GAT ATT GAT GTT GGA TGT2544 ACA GAC TTA AAT GAG GAC TTA GGT GTA TGG GTG ATA TTC AAG ATT AAG2592 ACG CAA GAT GGC CAT GCA AGA CTA GGG AAT CTA GAG TTT CTC GAA GAG2640 AAA CCA TTA TTA GGG GAA GCA CTA GCT CGT GTG AAA AGA GCG GAG AAG2688 AAG TGG AGA GAC AAA CGA GAG AAA CTG CAG TTG GAA ACA AAT ATT GTT2736 TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA2784 TAT GAT AGA TTA CAA GTG GAT ACG AAC ATC GCA ATG ATT CAT GCG GCA2832 GAT AAA CGC GTT CAT AGA ATC CGG GAA GCG TAT CTG CCA GAG TTG TCT2880 GTG ATT CCA GGT GTC AAT GCG GCC ATT TTC GAA GAA TTA GAG GGA CGT2928 ATT TTT ACA GCG TAT TCC TTA TAT GAT GCG AGA AAT GTC ATT AAA AAT2976 GGC GAT TTC AAT AAT GGC TTA TTA TGC TGG AAC GTG AAA GGT CAT GTA3024 GAT GTA GAA GAG CAA AAC AAC CAC CGT TCG GTC CTT GTT ATC CCA GAA3072 TGG GAG GCA GAA GTG TCA CAA GAG GTT CGT GTC TGT CCA GGT CGT GGC3120 TAT ATC CTT CGT GTC ACA GCA TAT AAA GAG GGA TAT GGA GAG GGC TGC3168 GTA ACG ATC CAT GAG ATC GAA GAC AAT ACA GAC GAA CTG AAA TTC AGC3216 AAC TGT GTA GAA GAG GAA GTA TAT CCA AAC AAC ACA GTA ACG TGT AAT3264 AAT TAT ACT GGG ACT CAA GAA GAA TAT GAG GGT ACG TAC ACT TCT CGT3312 AAT CAA GGA TAT GAC GAA GCC TAT GGT AAT AAC CCT TCC GTA CCA GCT3360 GAT TAC GCT TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA3408 GAG AAT CCT TGT GAA TCT AAC AGA GGC TAT GGG GAT TAC ACA CCA CTA3456 CCG GCT GGT TAT GTA ACA AAG GAT TTA GAG TAC TTC CCA GAG ACC GAT3504 AAG GTA TGG ATT GAG ATC GGA GAA ACA GAA GGA ACA TTC ATC GTG GAT3552 AGC GTG GAA TTA CTC CTT ATG GAG GAA 3579

7. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

U.S. Pat. No. 4,554,101

U.S. Pat. No. 4,683,195

U.S. Pat. No. 4,683,202

U.S. Pat. No. 4,702,914

U.S. Pat. No. 4,757,011

U.S. Pat. No. 4,769,061

U.S. Pat. No. 4,940,835

U.S. Pat. No. 4,965,188

U.S. Pat. No. 4,971,908

U.S. Pat. No. 5,004,863

U.S. Pat. No. 5,015,580

U.S. Pat. No. 5,055,294

U.S. Pat. No. 5,128,130

U.S. Pat. No. 5,176,995

U.S. Pat. No. 5,349,124

U.S. Pat. No. 5,380,831

U.S. Pat. No. 5,384,253

U.S. Pat. No. 5,416,102

U.S. Pat. No. 5,441,884

U.S. Pat. No. 5,449,681

U.S. Pat. No. 5,500,365.

Intl. Pat. Appl. Publ. No. WO 91/10725, published Jul. 25, 1991.

Intl. Pat. Appl. Publ. No. WO 93/07278, published Apr. 15, 1993.

Intl. Pat. Appl. Publ. No. WO 95/02058, published Jan. 19, 1995.

Intl. Pat. Appl. Publ. No. WO 95/06730, published Mar. 9, 1995.

Intl. Pat. Appl. Publ. No. WO 95/30752, published Nov. 16, 1995.

Intl. Pat. Appl. Publ. No. WO 95/30753, published Nov. 16, 1995.

Abdullah et al., Biotechnology, 4:1087, 1986.

Adelman et al., DNA, 2/3:183-193, 1983.

Allen and Choun, “Large unicellular liposomes with low uptake into thereticuloendothelial system,” FEBS Lett., 223:42-46, 1987.

Altschul, Stephen F. et al., “Basic local alignment search tool,” J.Mol. Biol., 215:403-410, 1990.

Arvidson et al., Mol. Biol., 3:1533-1534, 1989.

Baum et al., Appl. Environ. Microbial., 56:3420-3428, 1990.

Benbrook et al., In: Proceedings Bio Expo 1986, Butterworth, Stoneham,Mass., pp. 27-54, 1986.

Bolivar et al., Gene, 2:95, 1977.

Bytebier et al., Proc. Natl. Acad. Sci. USA, 84:5345, 1987.

Callis et al., Genes and Development, 1:1183, 1987.

Campbell, “Monoclonal Antibody Technology, Laboratory Techniques inBiochemistry and Molecular Biology,” Vol. 13, Burden and VonKnippenberg, Eds. pp. 75-83, Elsevier, Amsterdam, 1984.

Capecchi, M. R., “High efficiency transformation by directmicroinjection of DNA into cultured mammalian cells,” Cell22(2):479-488, 1980.

Cashmore et al., Gen. Eng. of Plants, Plenum Press, N.Y., 29-38, 1983.

Chambers et al., J. Bacterial., 173:3966-3976, 1991.

Chang et al., Nature, 375:615, 1978.

Chau et al., Science, 244:174-181, 1989.

Clapp, D. W., “Somatic gene therapy into hematopoietic cells. Currentstatus and future implications,” Clin. Perinatol. 20(1):155-168, 1993.

Couvreur et al., “Nanocapsules, a new lysosomotropic carrier,” FEBSLett., 84:323-326, 1977.

Couvreur, “Polyalkyleyanoacrylates as colloidal drug carriers,” Crit.Rev. Ther. Drug Carrier Syst., 5:1-20, 1988.

Crickmore et al., Abstr. 28th Annu. Meet. Soc. Invert. Pathol., CornellUniversity, Ithaca, N.Y., 1995.

Cristou et al., Plant Physiol, 87:671-674, 1988.

Curiel, D. T., Agarwal, S., Wagner, E., and Cotten, M., “Adenovirusenhancement of transferring-polylysine-mediated gene delivery,” Proc.Natl. Acad. Sci. USA 88(19):8850-8854, 1991.

Curiel, D. T., Wagner, E., and Cotten, M., Birnstiel, M. L., Agarwal,S., Li, C. M., Loechel, S., and Hu, P. C. high-efficiency gene transfermediated by adenovirus coupled to DNA-polylysine complexes,” Hum. Gen.Ther. 3(2):147-154, 1992.

Dhir et al., Plant Cell Reports, 10:97, 1991.

Eglitis, M. A., and Anderson, W. F., “Retroviral vectors forintroduction of genes into mammalian cells,” Biolechniques 6(7):608-614,1988.

Eglitis, M. A., Kantoff, P. W., Kohn, D. B., Karson, E., Moen, R. C.,Lothrop, C. D., Blaese, R. M., and Anderson, W. F., “Retroviral-mediatedgene transfer into hemopoietic cells,” Adv. Exp. Med. Biol. 241:19-27,1988.

Eichenlaub, R., J. Bacterial., 138(2):559-566, 1979.

Fiers et al., Nature, 273:113, 1978.

Fraley et al., Biotechnology, 3:629, 1985.

Fraley et al., Proc. Natl Acad. Sci. USA, 80:4803, 1983.

Fromm, M., Taylor, L. P., and Walbot, V., “Expression of genestransferred into monocot and dicot plant cells by electroporation,”Proc. Natl. Acad. Sci. USA 82(17):5824-5828, 1985.

Fujimura et al., Plant Tissue Culture Letters, 2:74, 1985.

Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J.C., and Robinson, H. L., “DNA vaccines: protective immunizations byparenteral, mucosal, and gene gun inoculations,” Proc. Natl. Acad. Sci.USA 90(24):11478-11482, 1993.

Gawron-Burke and Baum, Genet Engineer., 13:237-263, 1991.

Gefter et al., Somat. Cell Genet., 3:231-236, 1977.

Gill et al., J. Biol. Chem., 270:27277-27282, 1995.

Goding, “Monoclonal Antibodies: Principles and Practice,” pp. 60-74. 2ndEdition, Academic Press, Orlando, Fla., 1986.

Goeddel et al., Nature, 281:544, 1979.

Goeddel et al., Nucl. Acids Res., 8:4057, 1980.

Graham, F. L., and van der Eb, A. J., “Transformation of rat cells byDNA of human adenovirus 5,” Virology 54(2):536-539, 1973.

Green, Nucl. Acids Res. 16(1):369. 1988.

Grochulski et al., J. Mol. Biol., 254:447-464, 1995.

Harlow, E. and Lane, D. “Antibodies: A Laboratory Manual,” Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1988.

Harlow, E. and Lane, D. “Antibodies: A Laboratory Manual,” Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1988.

Henry-Michelland et al., “Attachment of antibiotics to nanoparticles;Preparation, drug-release and antimicrobial activity in vitro, Int. J.Pharm., 35:121-127, 1987.

Hess et al., J. Adv. Enzyme Reg., 7:149, 1968.

Hilber, U. W., Bodmer, M., Smith, F. D., and Koller, W., “Biolistictransformation of conidia of Botryotinia fuckeliana,” Curr. Genet.25(2):124-127, 1994.

Hitzeman et al., J. Biol. Chem., 255:2073, 1980.

Höfte and Whiteley, Microbial. Rev., 53:242-255, 1989.

Holland et al., Biochemistry, 17:4900, 1978.

Honee et al., Mol. Microbial., 5:2799-2806, 1991.

Hoover et al., (Eds.), “Remington's Pharmaceutical Sciences,” 15thEdition, Mack Publishing Co., Easton, Pa., 1975.

Horsch et al., Science, 227:1229-1231, 1985.

Horton et al., Gene, 77:61-68, 1989.

Humason, “Animal Tissue Techniques,” W.H. Freeman and Co., 1967.

Itakura et al., Science, 198:1056, 1977.

Jameson and Wolf, “The Antigenic Index: A Novel Algorithm for PredictingAntigenic Determinants,” Compu. Appl. Biosci., 4(1):181-6, 1988.

Johnston, S. A., and Tang, D. C., “Gene gun transfection of animal cellsand genetic immunization,” Methods Cell. Biol. 43(A):353-365, 1994.

Jones, Genetics, 85:12 1977.

Jorgensen et al., Mol. Gen. Genet., 207:471, 1987.

Keller et al., EMBO J., 8:1309-14, 1989.

Kingsman et al., Gene, 7:141, 1979.

Klee et al., Bio/Technology, 3:637, 1985.

Klein et al., Nature, 327:70, 1987.

Klein et al., Proc. Natl. Acad. Sci. USA, 85:8502-8505, 1988.

Knight et al., J. Biol. Chem., 270:17765-17770, 1995.

Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976.

Kohler and Milstein, Nature 256:495-497, 1975.

Krzyzek, R. A., “Genetic transformation of maize cells byelectroporation of cells pretreated with pectin degrading enzymes,” U.S.Pat. No. 5,384,253, Jan. 24, 1995.

Kuby, J., Immunology 2nd Edition, W.H. Freeman & Company, NY, 1994.

Kyte, J., and Doolittle, R. F., A simple method for displaying thehydropathic character of a protein,” J. Mol. Biol. 157(1):105-132, 1982.

Langridge et al., Proc. Natl. Acad. Sci. USA, 86:3219-3223, 1989.

Lee et al., Biochem. Biophys. Res. Comm., 216:306-312, 1995.

Lindstrom et al., Developmental Genetics, 11:160, 1990.

Lorz et al., Mol. Gen. Genet., 199:178, 1985.

Lu, L., Xiao, M., Clapp, D. W., Li, Z. H., and Broxmeyer, H. E., “Highefficiency retroviral mediated gene transduction into single isolatedimmature and replatable CD34(3+) hematopoietic sten/progenitor cellsfrom human umbilical cord blood,” J. Exp. Med. 178(6):2089-2096, 1993.

Luo et al., Plant Mol. Biol. Report., 6:165, 1988.

Maddock et al., Third Intl. Congr. Plant Mol. Biol., Abstr. No. 372,1991.

Maloy et al., “Microbial Genetics” 2nd Ed., Jones & Bartlett Publishers,Boston, Mass., 1994.

Maloy, S. R., “Experimental Techniques in Bacterial Genetics” Jones andBartlett Prokop, A., and Bajpai, R. K. “Recombinant DNA Technology I”Ann. N.Y. Acad. Sci. vol. 646, 1991.

Maniatis et al., “Molecular Cloning: a Laboratory Manual,” Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1982.

Marcotte et al., Nature, 335:454, 1988.

Masson et al., J. Biol. Chem., 270:20309-20315, 1995.

McCabe et al., Biotechnology, 6:923, 1988.

Mettus and Macaluso, Appl. Environ. Microbial., 56:1128-1134, 1990.

Nakamura et al., (1987) Enzyme Immunoassays: Heterogeneous andHomogeneous Systems, Chapter 27.

Neuhaus et al., Theor. Appl. Genet., 75:30, 1987.

Odell et al., Nature, 313:810, 1985.

Omirulleh et al., Plant Mol. Biol., 21:415-428, 1993.

Pena et al., Nature, 325:274, 1987.

Poszkowski et al., EMBO J., 3:2719, 1989.

Potrykus et al., Mol. Gen. Genel., 199:183, 1985.

Poulsen et al., Mol. Gen. Genet., 205:193-200, 1986.

Prokop, A., Bajpai, R. K., Ann. N.Y. Acad. Sci. 646, 1991.

Rogers et al., In: “Methods For Plant Molecular Biology,” A. Weissbachand H. Weissbach, eds., Academic Press Inc., San Diego, Calif. 1988.

Rogers et al., Methods Enzymol., 153:253-277, 1987.

Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

Schnepf et al., J. Biol. Chem., 265:20923-20930, 1990.

Segal, I. H., “Biochemical Calculations” 2nd Ed., John Wiley & Sons, NewYork, 1976.

Simpson, Science, 233:34, 1986.

Spielmann et al., Mol. Gen. Genet., 205:34, 1986.

Spoerel, Methods Enzymol., 152:588-597, 1987.

Stinchcomb et al., Nature, 282:39, 1979.

Thompson et al., Genet. Engineer., 17:99-117, 1995.

Toriyama et al., Theor. Appl. Genet., 73:16, 1986.

Tschemper et al., Gene, 10:157, 1980.

Uchimiya et al., Mol. Gen. Genet., 204:204, 1986.

Van Tunen et al., EMBO J., 7:1257, 1988.

Vasil et al., “Herbicide-resistant fertile transgenic wheat plantsobtained by microprojectile bombardment of regenerable embryogeniccallous,” Biotechnology, 10:667-674, 1992.

Vasil, Biotechnology, 6:397, 1988.

Vodkin et al., Cell, 34:1023, 1983.

Vogel et al., J. Cell Biochem., (Suppl.) 13D:312, 1989.

Wagner, E., Zatloukal, K., Cotten, M., Kirlappos, H., Mechtler, K.,Curiel, D. T., and Birnstiel, M. L., “Coupling of adenovirus totransferring-polylysine/DNA complexes greatly enhances receptor-mediatedgene delivery and expression of transfected genes,” Proc. Natl. Acad.Sci. USA 89(13):6099-6103, 1992.

Weissbach and Weissbach, Methods for Plant Molecular Biology, (eds.),Academic Press, Inc., San Diego, Calif., 1988.

Wenzler et al., Plant Mol. Biol., 12:41-50, 1989.

Wolf et al., “An Integrated Family of Amino Acid Sequence AnalysisPrograms,” Compu. Appl. Biosci., 4(1):187-91, 1988.

Wong, T. E., and Neumann, E., “Electric field mediated gene transfer,”Biochim. Biophys. Res. Commun., 107(2):584-587, 1982.

Yamada et al., Plant Cell Rep., 4:85, 1986.

Yang et al., Proc. Natl. Acad. Sci. USA, 87:4144-48, 1990.

Zhou et al., Methods Enzymol., 101:433, 1983.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

30 23 base pairs nucleic acid single linear not provided 1 GGATAGCACTCATCAAAGGT ACC 23 27 base pairs nucleic acid single linear not provided2 GAAGATATCC AATTCGAACA GTTTCCC 27 28 base pairs nucleic acid singlelinear not provided 3 CATATTCTGC CTCGAGTGTT GCAGTAAC 28 17 base pairsnucleic acid single linear not provided 4 CCCGATCGGC CGCATGC 17 17 basepairs nucleic acid single linear not provided 5 CATTGGAGCT CTCCATG 17 16base pairs nucleic acid single linear not provided 6 GCACTACGAT GTATCC16 20 base pairs nucleic acid single linear not provided 7 CATCGTAGTGCAACTCTTAC 20 39 base pairs nucleic acid single linear not provided 8CCAAGAAAAT ACTAGAGCTC TTGTTAAAAA AGGTGTTCC 39 3531 base pairs nucleicacid single linear not provided CDS 1..3531 9 ATG GAT AAC AAT CCG AACATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 Met Asp Asn Asn Pro Asn IleAsn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 Ser Asn Pro Glu Val Glu ValLeu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 Tyr Thr Pro Ile Asp Ile Ser LeuSer Leu Thr Gln Phe Leu Leu Ser 35 40 45 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 Glu Phe Val Pro Gly Ala Gly Phe ValLeu Gly Leu Val Asp Ile Ile 50 55 60 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp AspAla Phe Leu Val Gln Ile 65 70 75 80 GAA CAG TTA ATT AAC CAA AGA ATA GAAGAA TTC GCT AGG AAC CAA GCC 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu GluPhe Ala Arg Asn Gln Ala 85 90 95 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTTTAT CAA ATT TAC GCA GAA 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu TyrGln Ile Tyr Ala Glu 100 105 110 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACTAAT CCA GCA TTA AGA GAA 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr AsnPro Ala Leu Arg Glu 115 120 125 GAG ATG CGT ATT CAA TTC AAT GAC ATG AACAGT GCC CTT ACA ACC GCT 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn SerAla Leu Thr Thr Ala 130 135 140 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAAGTT CCT CTT TTA TCA GTA 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln ValPro Leu Leu Ser Val 145 150 155 160 TAT GTT CAA GCT GCA AAT TTA CAT TTATCA GTT TTG AGA GAT GTT TCA 528 Tyr Val Gln Ala Ala Asn Leu His Leu SerVal Leu Arg Asp Val Ser 165 170 175 GTG TTT GGA CAA AGG TGG GGA TTT GATGCC GCG ACT ATC AAT AGT CGT 576 Val Phe Gly Gln Arg Trp Gly Phe Asp AlaAla Thr Ile Asn Ser Arg 180 185 190 TAT AAT GAT TTA ACT AGG CTT ATT GGCAAC TAT ACA GAT TAT GCT GTA 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly AsnTyr Thr Asp Tyr Ala Val 195 200 205 CGC TGG TAC AAT ACG GGA TTA GAA CGTGTA TGG GGA CCG GAT TCT AGA 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg ValTrp Gly Pro Asp Ser Arg 210 215 220 GAT TGG GTA AGG TAT AAT CAA TTT AGAAGA GAA TTA ACA CTA ACT GTA 720 Asp Trp Val Arg Tyr Asn Gln Phe Arg ArgGlu Leu Thr Leu Thr Val 225 230 235 240 TTA GAT ATC GTT GCT CTG TTC CCGAAT TAT GAT AGT AGA AGA TAT CCA 768 Leu Asp Ile Val Ala Leu Phe Pro AsnTyr Asp Ser Arg Arg Tyr Pro 245 250 255 ATT CGA ACA GTT TCC CAA TTA ACAAGA GAA ATT TAT ACA AAC CCA GTA 816 Ile Arg Thr Val Ser Gln Leu Thr ArgGlu Ile Tyr Thr Asn Pro Val 260 265 270 TTA GAA AAT TTT GAT GGT AGT TTTCGA GGC TCG GCT CAG GGC ATA GAA 864 Leu Glu Asn Phe Asp Gly Ser Phe ArgGly Ser Ala Gln Gly Ile Glu 275 280 285 AGA AGT ATT AGG AGT CCA CAT TTGATG GAT ATA CTT AAC AGT ATA ACC 912 Arg Ser Ile Arg Ser Pro His Leu MetAsp Ile Leu Asn Ser Ile Thr 290 295 300 ATC TAT ACG GAT GCT CAT AGG GGTTAT TAT TAT TGG TCA GGG CAT CAA 960 Ile Tyr Thr Asp Ala His Arg Gly TyrTyr Tyr Trp Ser Gly His Gln 305 310 315 320 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 Ile Met Ala Ser Pro Val Gly PheSer Gly Pro Glu Phe Thr Phe Pro 325 330 335 CTA TAT GGA ACT ATG GGA AATGCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 Leu Tyr Gly Thr Met Gly Asn AlaAla Pro Gln Gln Arg Ile Val Ala 340 345 350 CAA CTA GGT CAG GGC GTG TATAGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 Gln Leu Gly Gln Gly Val Tyr ArgThr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 AGA CCT TTT AAT ATA GGG ATAAAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 Arg Pro Phe Asn Ile Gly Ile AsnAsn Gln Gln Leu Ser Val Leu Asp 370 375 380 GGG ACA GAA TTT GCT TAT GGAACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 Gly Thr Glu Phe Ala Tyr Gly ThrSer Ser Asn Leu Pro Ser Ala Val 385 390 395 400 TAC AGA AAA AGC GGA ACGGTA GAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 Tyr Arg Lys Ser Gly Thr ValAsp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 AAT AAC AAC GTG CCA CCTAGG CAA GGA TTT AGT CAT CGA TTA AGC CAT 1296 Asn Asn Asn Val Pro Pro ArgGln Gly Phe Ser His Arg Leu Ser His 420 425 430 GTT TCA ATG TTT CGT TCAGGC TTT AGT AAT AGT AGT GTA AGT ATA ATA 1344 Val Ser Met Phe Arg Ser GlyPhe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 AGA GCT CCA ATG TTT TCTTGG ACG CAC CGT AGT GCA ACC CCT ACA AAT 1392 Arg Ala Pro Met Phe Ser TrpThr His Arg Ser Ala Thr Pro Thr Asn 450 455 460 ACA ATT GAT CCG GAG AGGATT ACT CAA ATA CCA TTG GTA AAA GCA CAT 1440 Thr Ile Asp Pro Glu Arg IleThr Gln Ile Pro Leu Val Lys Ala His 465 470 475 480 ACA CTT CAG TCA GGTACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA 1488 Thr Leu Gln Ser Gly ThrThr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 GGA GAT ATT CTT CGACGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT 1536 Gly Asp Ile Leu Arg ArgThr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 GTT AAT ATA AAT GGGCAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC 1584 Val Asn Ile Asn Gly GlnLeu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 TAT GCC TCT ACT ACAAAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA 1632 Tyr Ala Ser Thr Thr AsnLeu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 CGG ATT TTT GCT GGTCAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA 1680 Arg Ile Phe Ala Gly GlnPhe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 TTA ACA TTC CAATCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA 1728 Leu Thr Phe Gln SerPhe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 TTC CCA ATG AGCCAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT 1776 Phe Pro Met Ser GlnSer Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 TCA GGG AAT GAAGTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT 1824 Ser Gly Asn Glu ValTyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 GCA ACA TTT GAAGCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG 1872 Ala Thr Phe Glu AlaGlu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AAT GCG CTG TTTACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG 1920 Asn Ala Leu Phe ThrSer Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635 640 ACG GAT TATCAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA 1968 Thr Asp Tyr HisIle Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645 650 655 GAT GAA TTTTGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA 2016 Asp Glu Phe CysLeu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 CAT GCG AAGCGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC 2064 His Ala Lys ArgLeu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685 TTC AAA GGCATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG 2112 Phe Lys Gly IleAsn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700 GAT ATT ACCATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC 2160 Asp Ile Thr IleGln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 ACA CTACCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA 2208 Thr Leu ProGly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 AAA ATCGAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA 2256 Lys Ile AspGlu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 GGG TATATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC 2304 Gly Tyr IleGlu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 AAT GCAAAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG 2352 Asn Ala LysHis Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 CCG CTTTCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA 2400 Pro Leu SerAla Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 TGCGCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG 2448 Cys AlaPro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 GATGGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT 2496 Asp GlyGlu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 GATGTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC 2544 Asp ValGly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 TTTAAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG 2592 Phe LysIle Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 TTTCTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA 2640 Phe LeuGlu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA 2688 ArgAla Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT 2736 ThrAsn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG 2784 ValAsn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG 2832 IleHis Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA 2880 ProGlu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955960 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT 2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970975 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG 2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985990 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT 3024Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 10001005 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys1010 1015 1020 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAGGGA TAT 3120 Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu GlyTyr 1025 1030 1035 1040 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AACAAT ACA GAC GAA 3168 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn AsnThr Asp Glu 1045 1050 1055 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATCTAT CCA AAT AAC ACG 3216 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile TyrPro Asn Asn Thr 1060 1065 1070 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAAGAA GAA TAC GGA GGT GCG 3264 Val Thr Cys Asn Asp Tyr Thr Val Asn Gln GluGlu Tyr Gly Gly Ala 1075 1080 1085 TAC ACT TCT CGT AAT CGA GGA TAT AACGAA GCT CCT TCC GTA CCA GCT 3312 Tyr Thr Ser Arg Asn Arg Gly Tyr Asn GluAla Pro Ser Val Pro Ala 1090 1095 1100 GAT TAT GCG TCA GTC TAT GAA GAAAAA TCG TAT ACA GAT GGA CGA AGA 3360 Asp Tyr Ala Ser Val Tyr Glu Glu LysSer Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 GAG AAT CCT TGT GAA TTTAAC AGA GGG TAT AGG GAT TAC ACG CCA CTA 3408 Glu Asn Pro Cys Glu Phe AsnArg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 CCA GTT GGT TAT GTGACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT 3456 Pro Val Gly Tyr Val ThrLys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 1145 1150 AAG GTA TGG ATTGAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC 3504 Lys Val Trp Ile GluIle Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1155 1160 1165 AGC GTG GAATTA CTC CTT ATG GAG GAA 3531 Ser Val Glu Leu Leu Leu Met Glu Glu 11701175 1177 amino acids amino acid linear protein not provided 10 Met AspAsn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 SerAsn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 TyrThr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 GluPhe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 TrpGly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu SerVal 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu ArgAsp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala ThrIle Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn TyrThr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg ValTrp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe ArgArg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu PhePro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser GlnLeu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe AspGly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile ArgSer Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr ThrAsp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 IleMet Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser AlaVal 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu IlePro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser HisArg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn SerSer Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr His ArgSer Ala Thr Pro Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Thr GlnIle Pro Leu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr ThrVal Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg ArgThr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn GlyGln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser ThrThr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile PheAla Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 LeuThr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595600 605 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val610 615 620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr AspVal 625 630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val AspCys Leu Ser 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu SerGlu Lys Val Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn LeuLeu Gln Asp Pro Asn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp ArgGly Trp Arg Gly Ser Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp AspVal Phe Lys Glu Asn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe AspGlu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser LysLeu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu AspSer Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys HisGlu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu SerAla Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 CysAla Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835840 845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu850 855 860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg ValLys 865 870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys LeuGlu Trp Glu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser ValAsp Ala Leu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala AspThr Asn Ile Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val His SerIle Arg Glu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly ValAsn Ala Ala Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe ThrAla Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly AspPhe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val AspVal Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val ProGlu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys 1010 1015 1020 ProGly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 1025 10301035 1040 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr AspGlu 1045 1050 1055 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr ProAsn Asn Thr 1060 1065 1070 Val Thr Cys Asn Asp Tyr Thr Val Asn Gln GluGlu Tyr Gly Gly Ala 1075 1080 1085 Tyr Thr Ser Arg Asn Arg Gly Tyr AsnGlu Ala Pro Ser Val Pro Ala 1090 1095 1100 Asp Tyr Ala Ser Val Tyr GluGlu Lys Ser Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 Glu Asn Pro CysGlu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 Pro ValGly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 1145 1150Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 11551160 1165 Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 3531 base pairsnucleic acid single linear not provided CDS 1..3531 11 ATG GAT AAC AATCCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 Met Asp Asn Asn ProAsn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 AGT AAC CCT GAAGTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 Ser Asn Pro Glu ValGlu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 TAC ACC CCA ATC GATATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 Tyr Thr Pro Ile Asp IleSer Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 GAA TTT GTT CCC GGT GCTGGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 Glu Phe Val Pro Gly Ala GlyPhe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 TGG GGA ATT TTT GGT CCC TCTCAA TGG GAC GCA TTT CTT GTA CAA ATT 240 Trp Gly Ile Phe Gly Pro Ser GlnTrp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 GAA CAG TTA ATT AAC CAA AGAATA GAA GAA TTC GCT AGG AAC CAA GCC 288 Glu Gln Leu Ile Asn Gln Arg IleGlu Glu Phe Ala Arg Asn Gln Ala 85 90 95 ATT TCT AGA TTA GAA GGA CTA AGCAAT CTT TAT CAA ATT TAC GCA GAA 336 Ile Ser Arg Leu Glu Gly Leu Ser AsnLeu Tyr Gln Ile Tyr Ala Glu 100 105 110 TCT TTT AGA GAG TGG GAA GCA GATCCT ACT AAT CCA GCA TTA AGA GAA 384 Ser Phe Arg Glu Trp Glu Ala Asp ProThr Asn Pro Ala Leu Arg Glu 115 120 125 GAG ATG CGT ATT CAA TTC AAT GACATG AAC AGT GCC CTT ACA ACC GCT 432 Glu Met Arg Ile Gln Phe Asn Asp MetAsn Ser Ala Leu Thr Thr Ala 130 135 140 ATT CCT CTT TTT GCA GTT CAA AATTAT CAA GTT CCT CTT TTA TCA GTA 480 Ile Pro Leu Phe Ala Val Gln Asn TyrGln Val Pro Leu Leu Ser Val 145 150 155 160 TAT GTT CAA GCT GCA AAT TTACAT TTA TCA GTT TTG AGA GAT GTT TCA 528 Tyr Val Gln Ala Ala Asn Leu HisLeu Ser Val Leu Arg Asp Val Ser 165 170 175 GTG TTT GGA CAA AGG TGG GGATTT GAT GCC GCG ACT ATC AAT AGT CGT 576 Val Phe Gly Gln Arg Trp Gly PheAsp Ala Ala Thr Ile Asn Ser Arg 180 185 190 TAT AAT GAT TTA ACT AGG CTTATT GGC AAC TAT ACA GAT TAT GCT GTA 624 Tyr Asn Asp Leu Thr Arg Leu IleGly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 CGC TGG TAC AAT ACG GGA TTAGAA CGT GTA TGG GGA CCG GAT TCT AGA 672 Arg Trp Tyr Asn Thr Gly Leu GluArg Val Trp Gly Pro Asp Ser Arg 210 215 220 GAT TGG GTA AGG TAT AAT CAATTT AGA AGA GAA TTA ACA CTA ACT GTA 720 Asp Trp Val Arg Tyr Asn Gln PheArg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 TTA GAT ATC GTT GCT CTGTTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 Leu Asp Ile Val Ala Leu PhePro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 ATT CGA ACA GTT TCC CAATTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 Ile Arg Thr Val Ser Gln LeuThr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 TTA GAA AAT TTT GAT GGTAGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 Leu Glu Asn Phe Asp Gly SerPhe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 AGA AGT ATT AGG AGT CCACAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 Arg Ser Ile Arg Ser Pro HisLeu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 ATC TAT ACG GAT GCT CATAGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 Ile Tyr Thr Asp Ala His ArgGly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ATA ATG GCT TCT CCTGTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 Ile Met Ala Ser Pro ValGly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 CTA TAT GGA ACT ATGGGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 Leu Tyr Gly Thr Met GlyAsn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 CAA CTA GGT CAG GGCGTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 Gln Leu Gly Gln Gly ValTyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 AGA CCT TTT AAT ATAGGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 Arg Pro Phe Asn Ile GlyIle Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 GGG ACA GAA TTT GCTTAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 Gly Thr Glu Phe Ala TyrGly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 TAC AGA AAA AGCGGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 Tyr Arg Lys Ser GlyThr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 AAT AAC AAC GTGCCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT 1296 Asn Asn Asn Val ProPro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 GTT TCA ATG TTTCGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA 1344 Val Ser Met Phe ArgSer Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 AGA GCT CCA ATGTTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT 1392 Arg Ala Pro Met PheSer Trp Thr His Arg Ser Ala Thr Pro Thr Asn 450 455 460 ACA ATT GAT CCGGAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT 1440 Thr Ile Asp Pro GluArg Ile Thr Gln Ile Pro Leu Val Lys Ala His 465 470 475 480 ACA CTT CAGTCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA 1488 Thr Leu Gln SerGly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 GGA GAT ATTCTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT 1536 Gly Asp Ile LeuArg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 GTT AAT ATAAAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC 1584 Val Asn Ile AsnGly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 TAT GCC TCTACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA 1632 Tyr Ala Ser ThrThr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 CGG ATT TTTGCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA 1680 Arg Ile Phe AlaGly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 TTA ACATTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA 1728 Leu Thr PheGln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 TTC CCAATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT 1776 Phe Pro MetSer Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 TCA GGGAAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT 1824 Ser Gly AsnGlu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 GCA ACACTC GAG GCT GAA TAT AAT CTG GAA AGA GCG CAG AAG GCG GTG 1872 Ala Thr LeuGlu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AAT GCGCTG TTT ACG TCT ACA AAC CAA CTA GGG CTA AAA ACA AAT GTA 1920 Asn Ala LeuPhe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val 625 630 635 640 ACGGAT TAT CAT ATT GAT CAA GTG TCC AAT TTA GTT ACG TAT TTA TCG 1968 Thr AspTyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser 645 650 655 GATGAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA 2016 Asp GluPhe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 CATGCG AAG CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT 2064 His AlaLys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn 675 680 685 TTCAAA GAC ATT AAT AGG CAA CCA GAA CGT GGG TGG GGC GGA AGT ACA 2112 Phe LysAsp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr 690 695 700 GGGATT ACC ATC CAA GGA GGG GAT GAC GTA TTT AAA GAA AAT TAC GTC 2160 Gly IleThr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720ACA CTA TCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA 2208 ThrLeu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA 2256 LysIle Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC 2304 GlyTyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG 2352 AsnAla Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA 2400 ProLeu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795800 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG 2448Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810815 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT 2496Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825830 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC 2544Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840845 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG 2592Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855860 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA 2640Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870875 880 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA2688 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885890 895 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT2736 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900905 910 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG2784 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915920 925 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG2832 Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930935 940 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA2880 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945950 955 960 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGAAAT 2928 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn965 970 975 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AACGTG 2976 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val980 985 990 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTCCTT 3024 Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu995 1000 1005 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGTGTC TGT 3072 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg ValCys 1010 1015 1020 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAGGAG GGA TAT 3120 Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys GluGly Tyr 1025 1030 1035 1040 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAGAAC AAT ACA GAC GAA 3168 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu AsnAsn Thr Asp Glu 1045 1050 1055 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAAATC TAT CCA AAT AAC ACG 3216 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu IleTyr Pro Asn Asn Thr 1060 1065 1070 GTA ACG TGT AAT GAT TAT ACT GTA AATCAA GAA GAA TAC GGA GGT GCG 3264 Val Thr Cys Asn Asp Tyr Thr Val Asn GlnGlu Glu Tyr Gly Gly Ala 1075 1080 1085 TAC ACT TCT CGT AAT CGA GGA TATAAC GAA GCT CCT TCC GTA CCA GCT 3312 Tyr Thr Ser Arg Asn Arg Gly Tyr AsnGlu Ala Pro Ser Val Pro Ala 1090 1095 1100 GAT TAT GCG TCA GTC TAT GAAGAA AAA TCG TAT ACA GAT GGA CGA AGA 3360 Asp Tyr Ala Ser Val Tyr Glu GluLys Ser Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 GAG AAT CCT TGT GAATTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA 3408 Glu Asn Pro Cys Glu PheAsn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 CCA GTT GGT TATGTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT 3456 Pro Val Gly Tyr ValThr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 1145 1150 AAG GTA TGGATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC 3504 Lys Val Trp IleGlu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1155 1160 1165 AGC GTGGAA TTA CTC CTT ATG GAG GAA 3531 Ser Val Glu Leu Leu Leu Met Glu Glu1170 1175 1177 amino acids amino acid linear protein not provided 12 MetAsp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 7580 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 9095 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr ThrAla 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu LeuSer Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val LeuArg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala AlaThr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly AsnTyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu ArgVal Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln PheArg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala LeuPhe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val SerGln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn PheAsp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser IleArg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile TyrThr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val LeuAsp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro SerAla Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp GluIle Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe SerHis Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser AsnSer Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr HisArg Ser Ala Thr Pro Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile ThrGln Ile Pro Leu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly ThrThr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu ArgArg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile AsnGly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala SerThr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg IlePhe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr595 600 605 Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys AlaVal 610 615 620 Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys ThrAsn Val 625 630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu ValThr Tyr Leu Ser 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu LeuSer Glu Lys Val Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg AsnLeu Leu Gln Asp Ser Asn 675 680 685 Phe Lys Asp Ile Asn Arg Gln Pro GluArg Gly Trp Gly Gly Ser Thr 690 695 700 Gly Ile Thr Ile Gln Gly Gly AspAsp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 Thr Leu Ser Gly Thr PheAsp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu SerLys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile GluAsp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala LysHis Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro LeuSer Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810815 Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820825 830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile835 840 845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn LeuGlu 850 855 860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala ArgVal Lys 865 870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu LysLeu Glu Trp Glu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu SerVal Asp Ala Leu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln AlaAsp Thr Asn Ile Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val HisSer Ile Arg Glu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro GlyVal Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile PheThr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn GlyAsp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His ValAsp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val ValPro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys 1010 1015 1020Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 10251030 1035 1040 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn ThrAsp Glu 1045 1050 1055 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile TyrPro Asn Asn Thr 1060 1065 1070 Val Thr Cys Asn Asp Tyr Thr Val Asn GlnGlu Glu Tyr Gly Gly Ala 1075 1080 1085 Tyr Thr Ser Arg Asn Arg Gly TyrAsn Glu Ala Pro Ser Val Pro Ala 1090 1095 1100 Asp Tyr Ala Ser Val TyrGlu Glu Lys Ser Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 Glu Asn ProCys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 ProVal Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 11451150 Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp1155 1160 1165 Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 3531 basepairs nucleic acid single linear not provided CDS 1..3531 13 ATG GAT AACAAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 Met Asp Asn AsnPro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 AGT AAC CCTGAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 Ser Asn Pro GluVal Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 TAC ACC CCA ATCGAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 GAA TTT GTT CCC GGTGCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 Glu Phe Val Pro Gly AlaGly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 TGG GGA ATT TTT GGT CCCTCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 Trp Gly Ile Phe Gly Pro SerGln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 GAA CAG TTA ATT AAC CAAAGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 Glu Gln Leu Ile Asn Gln ArgIle Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 ATT TCT AGA TTA GAA GGA CTAAGC AAT CTT TAT CAA ATT TAC GCA GAA 336 Ile Ser Arg Leu Glu Gly Leu SerAsn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 TCT TTT AGA GAG TGG GAA GCAGAT CCT ACT AAT CCA GCA TTA AGA GAA 384 Ser Phe Arg Glu Trp Glu Ala AspPro Thr Asn Pro Ala Leu Arg Glu 115 120 125 GAG ATG CGT ATT CAA TTC AATGAC ATG AAC AGT GCC CTT ACA ACC GCT 432 Glu Met Arg Ile Gln Phe Asn AspMet Asn Ser Ala Leu Thr Thr Ala 130 135 140 ATT CCT CTT TTT GCA GTT CAAAAT TAT CAA GTT CCT CTT TTA TCA GTA 480 Ile Pro Leu Phe Ala Val Gln AsnTyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 TAT GTT CAA GCT GCA AATTTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 Tyr Val Gln Ala Ala Asn LeuHis Leu Ser Val Leu Arg Asp Val Ser 165 170 175 GTG TTT GGA CAA AGG TGGGGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 Val Phe Gly Gln Arg Trp GlyPhe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 TAT AAT GAT TTA ACT AGGCTT ATT GGC AAC TAT ACA GAT CAT GCT GTA 624 Tyr Asn Asp Leu Thr Arg LeuIle Gly Asn Tyr Thr Asp His Ala Val 195 200 205 CGC TGG TAC AAT ACG GGATTA GAG CGT GTA TGG GGA CCG GAT TCT AGA 672 Arg Trp Tyr Asn Thr Gly LeuGlu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 GAT TGG ATA AGA TAT AATCAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 Asp Trp Ile Arg Tyr Asn GlnPhe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 TTA GAT ATC GTT TCTCTA TTT CCG AAC TAT GAT AGT AGA ACG TAT CCA 768 Leu Asp Ile Val Ser LeuPhe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro 245 250 255 ATT CGA ACA GTT TCCCAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 Ile Arg Thr Val Ser GlnLeu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 TTA GAA AAT TTT GATGGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 Leu Glu Asn Phe Asp GlySer Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 GGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 Gly Ser Ile Arg Ser ProHis Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 ATC TAT ACG GAT GCTCAT AGA GGA GAA TAT TAT TGG TCA GGG CAT CAA 960 Ile Tyr Thr Asp Ala HisArg Gly Glu Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ATA ATG GCT TCTCCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 CTA TAT GGA ACTATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 Leu Tyr Gly Thr MetGly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 CAA CTA GGT CAGGGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 Gln Leu Gly Gln GlyVal Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 AGA CCT TTT AATATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 Arg Pro Phe Asn IleGly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 GGG ACA GAA TTTGCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 Gly Thr Glu Phe AlaTyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 TAC AGA AAAAGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 Tyr Arg Lys SerGly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 AAT AAC AACGTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT 1296 Asn Asn Asn ValPro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 GTT TCA ATGTTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA 1344 Val Ser Met PheArg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 AGA GCT CCAATG TTT TCT TGG ACG CAC CGT AGT GCA ACC CCT ACA AAT 1392 Arg Ala Pro MetPhe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn 450 455 460 ACA ATT GATCCG GAG AGG ATT ACT CAA ATA CCA TTG GTA AAA GCA CAT 1440 Thr Ile Asp ProGlu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His 465 470 475 480 ACA CTTCAG TCA GGT ACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA 1488 Thr Leu GlnSer Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 GGA GATATT CTT CGA CGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT 1536 Gly Asp IleLeu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 GTT AATATA AAT GGG CAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC 1584 Val Asn IleAsn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 TAT GCCTCT ACT ACA AAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA 1632 Tyr Ala SerThr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 CGG ATTTTT GCT GGT CAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA 1680 Arg Ile PheAla Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 TTAACA TTC CAA TCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA 1728 Leu ThrPhe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 TTCCCA ATG AGC CAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT 1776 Phe ProMet Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 TCAGGG AAT GAA GTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT 1824 Ser GlyAsn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 GCAACA TTT GAA GCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG 1872 Ala ThrPhe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AATGCG CTG TTT ACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG 1920 Asn AlaLeu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635 640ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA 1968 ThrAsp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645 650 655GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA 2016 AspGlu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC 2064 HisAla Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685TTC AAA GGC ATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG 2112 PheLys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700GAT ATT ACC ATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC 2160 AspIle Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715720 ACA CTA CCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA 2208Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730735 AAA ATC GAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA 2256Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745750 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC 2304Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760765 AAT GCA AAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG 2352Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775780 CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA 2400Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790795 800 TGC GCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG2448 Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805810 815 GAT GGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT2496 Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820825 830 GAT GTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC2544 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835840 845 TTT AAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG2592 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850855 860 TTT CTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA2640 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865870 875 880 AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGGGAA 2688 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu885 890 895 ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTATTT 2736 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe900 905 910 GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCCATG 2784 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met915 920 925 ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TATCTG 2832 Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu930 935 940 CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAAGAA 2880 Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu945 950 955 960 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCGAGA AAT 2928 Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala ArgAsn 965 970 975 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGGAAC GTG 2976 Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp AsnVal 980 985 990 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCGGTC CTT 3024 Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser ValLeu 995 1000 1005 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTTCGT GTC TGT 3072 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val ArgVal Cys 1010 1015 1020 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TACAAG GAG GGA TAT 3120 Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr LysGlu Gly Tyr 1025 1030 1035 1040 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATCGAG AAC AAT ACA GAC GAA 3168 Gly Glu Gly Cys Val Thr Ile His Glu Ile GluAsn Asn Thr Asp Glu 1045 1050 1055 CTG AAG TTT AGC AAC TGC GTA GAA GAGGAA ATC TAT CCA AAT AAC ACG 3216 Leu Lys Phe Ser Asn Cys Val Glu Glu GluIle Tyr Pro Asn Asn Thr 1060 1065 1070 GTA ACG TGT AAT GAT TAT ACT GTAAAT CAA GAA GAA TAC GGA GGT GCG 3264 Val Thr Cys Asn Asp Tyr Thr Val AsnGln Glu Glu Tyr Gly Gly Ala 1075 1080 1085 TAC ACT TCT CGT AAT CGA GGATAT AAC GAA GCT CCT TCC GTA CCA GCT 3312 Tyr Thr Ser Arg Asn Arg Gly TyrAsn Glu Ala Pro Ser Val Pro Ala 1090 1095 1100 GAT TAT GCG TCA GTC TATGAA GAA AAA TCG TAT ACA GAT GGA CGA AGA 3360 Asp Tyr Ala Ser Val Tyr GluGlu Lys Ser Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 GAG AAT CCT TGTGAA TTT AAC AGA GGG TAT AGG GAT TAC ACG CCA CTA 3408 Glu Asn Pro Cys GluPhe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 CCA GTT GGTTAT GTG ACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT 3456 Pro Val Gly TyrVal Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 1145 1150 AAG GTATGG ATT GAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC 3504 Lys Val TrpIle Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1155 1160 1165 AGCGTG GAA TTA CTC CTT ATG GAG GAA 3531 Ser Val Glu Leu Leu Leu Met Glu Glu1170 1175 1177 amino acids amino acid linear protein not provided 14 MetAsp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 7580 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 9095 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr ThrAla 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu LeuSer Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val LeuArg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala AlaThr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly AsnTyr Thr Asp His Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu ArgVal Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Ile Arg Tyr Asn Gln PheArg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ser LeuPhe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro 245 250 255 Ile Arg Thr Val SerGln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn PheAsp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Gly Ser IleArg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile TyrThr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln 305 310 315 320Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val LeuAsp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro SerAla Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp GluIle Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe SerHis Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser AsnSer Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr HisArg Ser Ala Thr Pro Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile ThrGln Ile Pro Leu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly ThrThr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu ArgArg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile AsnGly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala SerThr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg IlePhe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr595 600 605 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys AlaVal 610 615 620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys ThrAsp Val 625 630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu ValAsp Cys Leu Ser 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu LeuSer Glu Lys Val Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg AsnLeu Leu Gln Asp Pro Asn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu AspArg Gly Trp Arg Gly Ser Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly AspAsp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr PheAsp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu SerLys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile GluAsp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala LysHis Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro LeuSer Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810815 Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820825 830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile835 840 845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn LeuGlu 850 855 860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala ArgVal Lys 865 870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu LysLeu Glu Trp Glu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu SerVal Asp Ala Leu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln AlaAsp Thr Asn Ile Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val HisSer Ile Arg Glu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro GlyVal Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile PheThr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn GlyAsp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His ValAsp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val ValPro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys 1010 1015 1020Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 10251030 1035 1040 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn ThrAsp Glu 1045 1050 1055 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile TyrPro Asn Asn Thr 1060 1065 1070 Val Thr Cys Asn Asp Tyr Thr Val Asn GlnGlu Glu Tyr Gly Gly Ala 1075 1080 1085 Tyr Thr Ser Arg Asn Arg Gly TyrAsn Glu Ala Pro Ser Val Pro Ala 1090 1095 1100 Asp Tyr Ala Ser Val TyrGlu Glu Lys Ser Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 Glu Asn ProCys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 ProVal Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 11451150 Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp1155 1160 1165 Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 20 basepairs nucleic acid single linear not provided 15 TATCCAATTC GAACGTCATC20 20 base pairs nucleic acid single linear not provided 16 TTTAGTCATCGATTAAATCA 20 20 base pairs nucleic acid single linear not provided 17ATAATAAGAG CTCCAATGTT 20 20 base pairs nucleic acid single linear notprovided 18 TACATCGTAG TGCAACTCTT 20 20 base pairs nucleic acid singlelinear not provided 19 TCATGGAGAG CTCCTATGTT 20 20 base pairs nucleicacid single linear not provided 20 TTAACAAGAG CTCCTATGTT 20 20 basepairs nucleic acid single linear not provided 21 ACTACCAGGT ACCTTTGATG20 20 base pairs nucleic acid single linear not provided 22 ACTACCGGGTACCTTTGATA 20 18 base pairs nucleic acid single linear not provided 23ATTTGAGTAA TACTATCC 18 19 base pairs nucleic acid single linear notprovided 24 ATTACTCAAA TACCATTGG 19 3534 base pairs nucleic acid singlelinear not provided CDS 1..3531 25 ATG GAT AAC AAT CCG AAC ATC AAT GAATGC ATT CCT TAT AAT TGT TTA 48 Met Asp Asn Asn Pro Asn Ile Asn Glu CysIle Pro Tyr Asn Cys Leu 1 5 10 15 AGT AAC CCT GAA GTA GAA GTA TTA GGTGGA GAA AGA ATA GAA ACT GGT 96 Ser Asn Pro Glu Val Glu Val Leu Gly GlyGlu Arg Ile Glu Thr Gly 20 25 30 TAC ACC CCA ATC GAT ATT TCC TTG TCG CTAACG CAA TTT CTT TTG AGT 144 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu ThrGln Phe Leu Leu Ser 35 40 45 GAA TTT GTT CCC GGT GCT GGA TTT GTG TTA GGACTA GTT GAT ATA ATA 192 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly LeuVal Asp Ile Ile 50 55 60 TGG GGA ATT TTT GGT CCC TCT CAA TGG GAC GCA TTTCTT GTA CAA ATT 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe LeuVal Gln Ile 65 70 75 80 GAA CAG TTA ATT AAC CAA AGA ATA GAA GAA TTC GCTAGG AAC CAA GCC 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala ArgAsn Gln Ala 85 90 95 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTT TAT CAA ATTTAC GCA GAA 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile TyrAla Glu 100 105 110 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACT AAT CCA GCATTA AGA GAA 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala LeuArg Glu 115 120 125 GAG ATG CGT ATT CAA TTC AAT GAC ATG AAC AGT GCC CTTACA ACC GCT 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu ThrThr Ala 130 135 140 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAA GTT CCT CTTTTA TCA GTA 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu LeuSer Val 145 150 155 160 TAT GTT CAA GCT GCA AAT TTA CAT TTA TCA GTT TTGAGA GAT GTT TCA 528 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu ArgAsp Val Ser 165 170 175 GTG TTT GGA CAA AGG TGG GGA TTT GAT GCC GCG ACTATC AAT AGT CGT 576 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr IleAsn Ser Arg 180 185 190 TAT AAT GAT TTA ACT AGG CTT ATT GGC AAC TAT ACAGAT CAT GCT GTA 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr AspHis Ala Val 195 200 205 CGC TGG TAC AAT ACG GGA TTA GAG CGT GTA TGG GGACCG GAT TCT AGA 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly ProAsp Ser Arg 210 215 220 GAT TGG ATA AGA TAT AAT CAA TTT AGA AGA GAA TTAACA CTA ACT GTA 720 Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 TTA GAT ATC GTT TCT CTA TTT CCG AAC TAT GATAGT AGA ACG TAT CCA 768 Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp SerArg Thr Tyr Pro 245 250 255 ATT CGA ACA GTT TCC CAA TTA ACA AGA GAA ATTTAT ACA AAC CCA GTA 816 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile TyrThr Asn Pro Val 260 265 270 TTA GAA AAT TTT GAT GGT AGT TTT CGA GGC TCGGCT CAG GGC ATA GAA 864 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser AlaGln Gly Ile Glu 275 280 285 AGA AGT ATT AGG AGT CCA CAT TTG ATG GAT ATACTT AAC AGT ATA ACC 912 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile LeuAsn Ser Ile Thr 290 295 300 ATC TAT ACG GAT GCT CAT AGG GGT TAT TAT TATTGG TCA GGG CAT CAA 960 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr TrpSer Gly His Gln 305 310 315 320 ATA ATG GCT TCT CCT GTA GGG TTT TCG GGGCCA GAA TTC ACT TTT CCG 1008 Ile Met Ala Ser Pro Val Gly Phe Ser Gly ProGlu Phe Thr Phe Pro 325 330 335 CTA TAT GGA ACT ATG GGA AAT GCA GCT CCACAA CAA CGT ATT GTT GCT 1056 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro GlnGln Arg Ile Val Ala 340 345 350 CAA CTA GGT CAG GGC GTG TAT AGA ACA TTATCG TCC ACT TTA TAT AGA 1104 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu SerSer Thr Leu Tyr Arg 355 360 365 AGA CCT TTT AAT ATA GGG ATA AAT AAT CAACAA CTA TCT GTT CTT GAC 1152 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln GlnLeu Ser Val Leu Asp 370 375 380 GGG ACA GAA TTT GCT TAT GGA ACC TCC TCAAAT TTG CCA TCC GCT GTA 1200 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser AsnLeu Pro Ser Ala Val 385 390 395 400 TAC AGA AAA AGC GGA ACG GTA GAT TCGCTG GAT GAA ATA CCG CCA CAG 1248 Tyr Arg Lys Ser Gly Thr Val Asp Ser LeuAsp Glu Ile Pro Pro Gln 405 410 415 AAT AAC AAC GTG CCA CCT AGG CAA GGATTT AGT CAT CGA TTA AGC CAT 1296 Asn Asn Asn Val Pro Pro Arg Gln Gly PheSer His Arg Leu Ser His 420 425 430 GTT TCA ATG TTT CGT TCA GGC TTT AGTAAT AGT AGT GTA AGT ATA ATA 1344 Val Ser Met Phe Arg Ser Gly Phe Ser AsnSer Ser Val Ser Ile Ile 435 440 445 AGA GCT CCA ATG TTT TCT TGG ACG CACCGT AGT GCA ACC CCT ACA AAT 1392 Arg Ala Pro Met Phe Ser Trp Thr His ArgSer Ala Thr Pro Thr Asn 450 455 460 ACA ATT GAT CCG GAG AGG ATT ACT CAAATA CCA TTG GTA AAA GCA CAT 1440 Thr Ile Asp Pro Glu Arg Ile Thr Gln IlePro Leu Val Lys Ala His 465 470 475 480 ACA CTT CAG TCA GGT ACT ACT GTTGTA AGA GGG CCC GGG TTT ACG GGA 1488 Thr Leu Gln Ser Gly Thr Thr Val ValArg Gly Pro Gly Phe Thr Gly 485 490 495 GGA GAT ATT CTT CGA CGA ACA AGTGGA GGA CCA TTT GCT TAT ACT ATT 1536 Gly Asp Ile Leu Arg Arg Thr Ser GlyGly Pro Phe Ala Tyr Thr Ile 500 505 510 GTT AAT ATA AAT GGG CAA TTA CCCCAA AGG TAT CGT GCA AGA ATA CGC 1584 Val Asn Ile Asn Gly Gln Leu Pro GlnArg Tyr Arg Ala Arg Ile Arg 515 520 525 TAT GCC TCT ACT ACA AAT CTA AGAATT TAC GTA ACG GTT GCA GGT GAA 1632 Tyr Ala Ser Thr Thr Asn Leu Arg IleTyr Val Thr Val Ala Gly Glu 530 535 540 CGG ATT TTT GCT GGT CAA TTT AACAAA ACA ATG GAT ACC GGT GAC CCA 1680 Arg Ile Phe Ala Gly Gln Phe Asn LysThr Met Asp Thr Gly Asp Pro 545 550 555 560 TTA ACA TTC CAA TCT TTT AGTTAC GCA ACT ATT AAT ACA GCT TTT ACA 1728 Leu Thr Phe Gln Ser Phe Ser TyrAla Thr Ile Asn Thr Ala Phe Thr 565 570 575 TTC CCA ATG AGC CAG AGT AGTTTC ACA GTA GGT GCT GAT ACT TTT AGT 1776 Phe Pro Met Ser Gln Ser Ser PheThr Val Gly Ala Asp Thr Phe Ser 580 585 590 TCA GGG AAT GAA GTT TAT ATAGAC AGA TTT GAA TTG ATT CCA GTT ACT 1824 Ser Gly Asn Glu Val Tyr Ile AspArg Phe Glu Leu Ile Pro Val Thr 595 600 605 GCA ACA TTT GAA GCA GAA TATGAT TTA GAA AGA GCA CAA AAG GCG GTG 1872 Ala Thr Phe Glu Ala Glu Tyr AspLeu Glu Arg Ala Gln Lys Ala Val 610 615 620 AAT GCG CTG TTT ACT TCT ATAAAC CAA ATA GGG ATA AAA ACA GAT GTG 1920 Asn Ala Leu Phe Thr Ser Ile AsnGln Ile Gly Ile Lys Thr Asp Val 625 630 635 640 ACG GAT TAT CAT ATT GATCAA GTA TCC AAT TTA GTG GAT TGT TTA TCA 1968 Thr Asp Tyr His Ile Asp GlnVal Ser Asn Leu Val Asp Cys Leu Ser 645 650 655 GAT GAA TTT TGT CTG GATGAA AAG CGA GAA TTG TCC GAG AAA GTC AAA 2016 Asp Glu Phe Cys Leu Asp GluLys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 CAT GCG AAG CGA CTC AGTGAT GAG CGG AAT TTA CTT CAA GAT CCA AAC 2064 His Ala Lys Arg Leu Ser AspGlu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685 TTC AAA GGC ATC AAT AGGCAA CTA GAC CGT GGT TGG AGA GGA AGT ACG 2112 Phe Lys Gly Ile Asn Arg GlnLeu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700 GAT ATT ACC ATC CAA AGAGGA GAT GAC GTA TTC AAA GAA AAT TAT GTC 2160 Asp Ile Thr Ile Gln Arg GlyAsp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 ACA CTA CCA GGT ACCTTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA 2208 Thr Leu Pro Gly Thr PheAsp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 AAA ATC GAT GAA TCAAAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA 2256 Lys Ile Asp Glu Ser LysLeu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 GGG TAT ATC GAA GATAGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC 2304 Gly Tyr Ile Glu Asp SerGln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 AAT GCA AAA CAT GAAACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG 2352 Asn Ala Lys His Glu ThrVal Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 CCG CTT TCA GCC CAAAGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA 2400 Pro Leu Ser Ala Gln SerPro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 TGC GCG CCA CACCTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG 2448 Cys Ala Pro His LeuGlu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 GAT GGA GAA AAGTGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT 2496 Asp Gly Glu Lys CysAla His His Ser His His Phe Ser Leu Asp Ile 820 825 830 GAT GTA GGA TGTACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC 2544 Asp Val Gly Cys ThrAsp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 TTT AAG ATT AAGACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG 2592 Phe Lys Ile Lys ThrGln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 TTT CTC GAA GAGAAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA 2640 Phe Leu Glu Glu LysPro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880 AGA GCG GAGAAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA 2688 Arg Ala Glu LysLys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895 ACA AAT ATCGTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT 2736 Thr Asn Ile ValTyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910 GTA AAC TCTCAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG 2784 Val Asn Ser GlnTyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925 ATT CAT GCGGCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG 2832 Ile His Ala AlaAsp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940 CCT GAG CTGTCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA 2880 Pro Glu Leu SerVal Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 TTA GAAGGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT 2928 Leu Glu GlyArg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 GTC ATTAAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG 2976 Val Ile LysAsn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 AAA GGGCAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT 3024 Lys Gly HisVal Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 GTTGTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT 3072 Val ValPro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys 1010 1015 1020CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAG GGA TAT 3120 ProGly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 1025 10301035 1040 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AAC AAT ACA GACGAA 3168 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu1045 1050 1055 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATC TAT CCA AATAAC ACG 3216 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn AsnThr 1060 1065 1070 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAA GAA GAA TACGGA GGT GCG 3264 Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr GlyGly Ala 1075 1080 1085 TAC ACT TCT CGT AAT CGA GGA TAT AAC GAA GCT CCTTCC GTA CCA GCT 3312 Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro SerVal Pro Ala 1090 1095 1100 GAT TAT GCG TCA GTC TAT GAA GAA AAA TCG TATACA GAT GGA CGA AGA 3360 Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr ThrAsp Gly Arg Arg 1105 1110 1115 1120 GAG AAT CCT TGT GAA TTT AAC AGA GGGTAT AGG GAT TAC ACG CCA CTA 3408 Glu Asn Pro Cys Glu Phe Asn Arg Gly TyrArg Asp Tyr Thr Pro Leu 1125 1130 1135 CCA GTT GGT TAT GTG ACA AAA GAATTA GAA TAC TTC CCA GAA ACC GAT 3456 Pro Val Gly Tyr Val Thr Lys Glu LeuGlu Tyr Phe Pro Glu Thr Asp 1140 1145 1150 AAG GTA TGG ATT GAG ATT GGAGAA ACG GAA GGA ACA TTT ATC GTG GAC 3504 Lys Val Trp Ile Glu Ile Gly GluThr Glu Gly Thr Phe Ile Val Asp 1155 1160 1165 AGC GTG GAA TTA CTC CTTATG GAG GAA TAG 3534 Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 1177amino acids amino acid linear protein not provided 26 Met Asp Asn AsnPro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn ProGlu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr ProIle Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe ValPro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly IlePhe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu GlnLeu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile SerArg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 SerPhe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp ValSer 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile AsnSer Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr AspHis Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp GlyPro Asp Ser Arg 210 215 220 Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg GluLeu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ser Leu Phe Pro AsnTyr Asp Ser Arg Thr Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu ThrArg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly SerPhe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser ProHis Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp AlaHis Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met AlaSer Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu TyrGly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 GlnLeu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro ProGln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg LeuSer His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser ValSer Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser AlaThr Pro Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile ProLeu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly Thr Thr Val ValArg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr SerGly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile Asn Gly Gln LeuPro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr AsnLeu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg Ile Phe Ala GlyGln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 Leu Thr PheGln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 Phe ProMet Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 SerGly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys LeuSer 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu LysVal Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu GlnAsp Pro Asn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly TrpArg Gly Ser Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly Asp Asp Val PheLys Glu Asn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr Phe Asp Glu CysTyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu Ser Lys Leu LysAla Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile Glu Asp Ser GlnAsp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala Lys His Glu ThrVal Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro Leu Ser Ala GlnSer Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 Cys Ala ProHis Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 Asp GlyGlu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 AspVal Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu TrpGlu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp AlaLeu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr AsnIle Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val His Ser Ile ArgGlu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro Gly Val Asn AlaAla Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile Phe Thr Ala PheSer Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn Gly Asp Phe AsnAsn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His Val Asp Val GluGlu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val Val Pro Glu TrpGlu Ala Glu Val Ser Gln Glu Val Arg Val Cys 1010 1015 1020 Pro Gly ArgGly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 1025 1030 1035 1040Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu 10451050 1055 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn AsnThr 1060 1065 1070 Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu TyrGly Gly Ala 1075 1080 1085 Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu AlaPro Ser Val Pro Ala 1090 1095 1100 Asp Tyr Ala Ser Val Tyr Glu Glu LysSer Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 Glu Asn Pro Cys Glu PheAsn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 Pro Val Gly TyrVal Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 1145 1150 Lys ValTrp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1155 1160 1165Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 3534 base pairs nucleicacid single linear not provided CDS 1..3531 27 ATG GAT AAC AAT CCG AACATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 Met Asp Asn Asn Pro Asn IleAsn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 AGT AAC CCT GAA GTA GAAGTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 Ser Asn Pro Glu Val Glu ValLeu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 TAC ACC CCA ATC GAT ATT TCCTTG TCG CTA ACG CAA TTT CTT TTG AGT 144 Tyr Thr Pro Ile Asp Ile Ser LeuSer Leu Thr Gln Phe Leu Leu Ser 35 40 45 GAA TTT GTT CCC GGT GCT GGA TTTGTG TTA GGA CTA GTT GAT ATA ATA 192 Glu Phe Val Pro Gly Ala Gly Phe ValLeu Gly Leu Val Asp Ile Ile 50 55 60 TGG GGA ATT TTT GGT CCC TCT CAA TGGGAC GCA TTT CTT GTA CAA ATT 240 Trp Gly Ile Phe Gly Pro Ser Gln Trp AspAla Phe Leu Val Gln Ile 65 70 75 80 GAA CAG TTA ATT AAC CAA AGA ATA GAAGAA TTC GCT AGG AAC CAA GCC 288 Glu Gln Leu Ile Asn Gln Arg Ile Glu GluPhe Ala Arg Asn Gln Ala 85 90 95 ATT TCT AGA TTA GAA GGA CTA AGC AAT CTTTAT CAA ATT TAC GCA GAA 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu TyrGln Ile Tyr Ala Glu 100 105 110 TCT TTT AGA GAG TGG GAA GCA GAT CCT ACTAAT CCA GCA TTA AGA GAA 384 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr AsnPro Ala Leu Arg Glu 115 120 125 GAG ATG CGT ATT CAA TTC AAT GAC ATG AACAGT GCC CTT ACA ACC GCT 432 Glu Met Arg Ile Gln Phe Asn Asp Met Asn SerAla Leu Thr Thr Ala 130 135 140 ATT CCT CTT TTT GCA GTT CAA AAT TAT CAAGTT CCT CTT TTA TCA GTA 480 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln ValPro Leu Leu Ser Val 145 150 155 160 TAT GTT CAA GCT GCA AAT TTA CAT TTATCA GTT TTG AGA GAT GTT TCA 528 Tyr Val Gln Ala Ala Asn Leu His Leu SerVal Leu Arg Asp Val Ser 165 170 175 GTG TTT GGA CAA AGG TGG GGA TTT GATGCC GCG ACT ATC AAT AGT CGT 576 Val Phe Gly Gln Arg Trp Gly Phe Asp AlaAla Thr Ile Asn Ser Arg 180 185 190 TAT AAT GAT TTA ACT AGG CTT ATT GGCAAC TAT ACA GAT TAT GCT GTA 624 Tyr Asn Asp Leu Thr Arg Leu Ile Gly AsnTyr Thr Asp Tyr Ala Val 195 200 205 CGC TGG TAC AAT ACG GGA TTA GAA CGTGTA TGG GGA CCG GAT TCT AGA 672 Arg Trp Tyr Asn Thr Gly Leu Glu Arg ValTrp Gly Pro Asp Ser Arg 210 215 220 GAT TGG GTA AGG TAT AAT CAA TTT AGAAGA GAA TTA ACA CTA ACT GTA 720 Asp Trp Val Arg Tyr Asn Gln Phe Arg ArgGlu Leu Thr Leu Thr Val 225 230 235 240 TTA GAT ATC GTT GCT CTG TTC CCGAAT TAT GAT AGT AGA AGA TAT CCA 768 Leu Asp Ile Val Ala Leu Phe Pro AsnTyr Asp Ser Arg Arg Tyr Pro 245 250 255 ATT CGA ACA GTT TCC CAA TTA ACAAGA GAA ATT TAT ACA AAC CCA GTA 816 Ile Arg Thr Val Ser Gln Leu Thr ArgGlu Ile Tyr Thr Asn Pro Val 260 265 270 TTA GAA AAT TTT GAT GGT AGT TTTCGA GGC TCG GCT CAG GGC ATA GAA 864 Leu Glu Asn Phe Asp Gly Ser Phe ArgGly Ser Ala Gln Gly Ile Glu 275 280 285 AGA AGT ATT AGG AGT CCA CAT TTGATG GAT ATA CTT AAC AGT ATA ACC 912 Arg Ser Ile Arg Ser Pro His Leu MetAsp Ile Leu Asn Ser Ile Thr 290 295 300 ATC TAT ACG GAT GCT CAT AGG GGTTAT TAT TAT TGG TCA GGG CAT CAA 960 Ile Tyr Thr Asp Ala His Arg Gly TyrTyr Tyr Trp Ser Gly His Gln 305 310 315 320 ATA ATG GCT TCT CCT GTA GGGTTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 Ile Met Ala Ser Pro Val Gly PheSer Gly Pro Glu Phe Thr Phe Pro 325 330 335 CTA TAT GGA ACT ATG GGA AATGCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 Leu Tyr Gly Thr Met Gly Asn AlaAla Pro Gln Gln Arg Ile Val Ala 340 345 350 CAA CTA GGT CAG GGC GTG TATAGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 Gln Leu Gly Gln Gly Val Tyr ArgThr Leu Ser Ser Thr Leu Tyr Arg 340 345 350 AGA CCT TTT AAT ATA GGG ATAAAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 Arg Pro Phe Asn Ile Gly Ile AsnAsn Gln Gln Leu Ser Val Leu Asp 370 375 380 GGG ACA GAA TTT GCT TAT GGAACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 Gly Thr Glu Phe Ala Tyr Gly ThrSer Ser Asn Leu Pro Ser Ala Val 385 390 395 400 TAC AGA AAA AGC GGA ACGGTA GAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 Tyr Arg Lys Ser Gly Thr ValAsp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 AAT AAC AAC GTG CCA CCTAGG CAA GGA TTT AGT CAT CGA TTA AGC CAT 1296 Asn Asn Asn Val Pro Pro ArgGln Gly Phe Ser His Arg Leu Ser His 420 425 430 GTT TCA ATG TTT CGT TCAGGC TTT AGT AAT AGT AGT GTA AGT ATA ATA 1344 Val Ser Met Phe Arg Ser GlyPhe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 AGA GCT CCT ATG TTC TCTTGG ATA CAT CGT AGT GCT GAA TTT AAT AAT 1392 Arg Ala Pro Met Phe Ser TrpIle His Arg Ser Ala Glu Phe Asn Asn 450 455 460 ATA ATT GCA TCG GAT AGTATT ACT CAA ATA CCA TTG GTA AAA GCA CAT 1440 Ile Ile Ala Ser Asp Ser IleThr Gln Ile Pro Leu Val Lys Ala His 465 470 475 480 ACA CTT CAG TCA GGTACT ACT GTT GTA AGA GGG CCC GGG TTT ACG GGA 1488 Thr Leu Gln Ser Gly ThrThr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 GGA GAT ATT CTT CGACGA ACA AGT GGA GGA CCA TTT GCT TAT ACT ATT 1536 Gly Asp Ile Leu Arg ArgThr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 GTT AAT ATA AAT GGGCAA TTA CCC CAA AGG TAT CGT GCA AGA ATA CGC 1584 Val Asn Ile Asn Gly GlnLeu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 TAT GCC TCT ACT ACAAAT CTA AGA ATT TAC GTA ACG GTT GCA GGT GAA 1632 Tyr Ala Ser Thr Thr AsnLeu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 CGG ATT TTT GCT GGTCAA TTT AAC AAA ACA ATG GAT ACC GGT GAC CCA 1680 Arg Ile Phe Ala Gly GlnPhe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560 TTA ACA TTC CAATCT TTT AGT TAC GCA ACT ATT AAT ACA GCT TTT ACA 1728 Leu Thr Phe Gln SerPhe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570 575 TTC CCA ATG AGCCAG AGT AGT TTC ACA GTA GGT GCT GAT ACT TTT AGT 1776 Phe Pro Met Ser GlnSer Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580 585 590 TCA GGG AAT GAAGTT TAT ATA GAC AGA TTT GAA TTG ATT CCA GTT ACT 1824 Ser Gly Asn Glu ValTyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr 595 600 605 GCA ACA TTT GAAGCA GAA TAT GAT TTA GAA AGA GCA CAA AAG GCG GTG 1872 Ala Thr Phe Glu AlaGlu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val 610 615 620 AAT GCG CTG TTTACT TCT ATA AAC CAA ATA GGG ATA AAA ACA GAT GTG 1920 Asn Ala Leu Phe ThrSer Ile Asn Gln Ile Gly Ile Lys Thr Asp Val 625 630 635 640 ACG GAT TATCAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA 1968 Thr Asp Tyr HisIle Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser 645 650 655 GAT GAA TTTTGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA 2016 Asp Glu Phe CysLeu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys 660 665 670 CAT GCG AAGCGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC 2064 His Ala Lys ArgLeu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn 675 680 685 TTC AAA GGCATC AAT AGG CAA CTA GAC CGT GGT TGG AGA GGA AGT ACG 2112 Phe Lys Gly IleAsn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700 GAT ATT ACCATC CAA AGA GGA GAT GAC GTA TTC AAA GAA AAT TAT GTC 2160 Asp Ile Thr IleGln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 ACA CTACCA GGT ACC TTT GAT GAG TGC TAT CCA ACA TAT TTG TAT CAA 2208 Thr Leu ProGly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 AAA ATCGAT GAA TCA AAA TTA AAA GCC TTT ACC CGT TAT CAA TTA AGA 2256 Lys Ile AspGlu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 GGG TATATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTA ATT CGC TAC 2304 Gly Tyr IleGlu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 AAT GCAAAA CAT GAA ACA GTA AAT GTG CCA GGT ACG GGT TCC TTA TGG 2352 Asn Ala LysHis Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 CCG CTTTCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAG CCG AAT CGA 2400 Pro Leu SerAla Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800 TGCGCG CCA CAC CTT GAA TGG AAT CCT GAC TTA GAT TGT TCG TGT AGG 2448 Cys AlaPro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810 815 GATGGA GAA AAG TGT GCC CAT CAT TCG CAT CAT TTC TCC TTA GAC ATT 2496 Asp GlyGlu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820 825 830 GATGTA GGA TGT ACA GAC TTA AAT GAG GAC CTA GGT GTA TGG GTG ATC 2544 Asp ValGly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile 835 840 845 TTTAAG ATT AAG ACG CAA GAT GGG CAC GCA AGA CTA GGG AAT CTA GAG 2592 Phe LysIle Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu 850 855 860 TTTCTC GAA GAG AAA CCA TTA GTA GGA GAA GCG CTA GCT CGT GTG AAA 2640 Phe LeuGlu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys 865 870 875 880AGA GCG GAG AAA AAA TGG AGA GAC AAA CGT GAA AAA TTG GAA TGG GAA 2688 ArgAla Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 885 890 895ACA AAT ATC GTT TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT 2736 ThrAsn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe 900 905 910GTA AAC TCT CAA TAT GAT CAA TTA CAA GCG GAT ACG AAT ATT GCC ATG 2784 ValAsn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met 915 920 925ATT CAT GCG GCA GAT AAA CGT GTT CAT AGC ATT CGA GAA GCT TAT CTG 2832 IleHis Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu 930 935 940CCT GAG CTG TCT GTG ATT CCG GGT GTC AAT GCG GCT ATT TTT GAA GAA 2880 ProGlu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu 945 950 955960 TTA GAA GGG CGT ATT TTC ACT GCA TTC TCC CTA TAT GAT GCG AGA AAT 2928Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970975 GTC ATT AAA AAT GGT GAT TTT AAT AAT GGC TTA TCC TGC TGG AAC GTG 2976Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985990 AAA GGG CAT GTA GAT GTA GAA GAA CAA AAC AAC CAA CGT TCG GTC CTT 3024Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 10001005 GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT3072 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys1010 1015 1020 CCG GGT CGT GGC TAT ATC CTT CGT GTC ACA GCG TAC AAG GAGGGA TAT 3120 Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu GlyTyr 1025 1030 1035 1040 GGA GAA GGT TGC GTA ACC ATT CAT GAG ATC GAG AACAAT ACA GAC GAA 3168 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn AsnThr Asp Glu 1045 1050 1055 CTG AAG TTT AGC AAC TGC GTA GAA GAG GAA ATCTAT CCA AAT AAC ACG 3216 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile TyrPro Asn Asn Thr 1060 1065 1070 GTA ACG TGT AAT GAT TAT ACT GTA AAT CAAGAA GAA TAC GGA GGT GCG 3264 Val Thr Cys Asn Asp Tyr Thr Val Asn Gln GluGlu Tyr Gly Gly Ala 1075 1080 1085 TAC ACT TCT CGT AAT CGA GGA TAT AACGAA GCT CCT TCC GTA CCA GCT 3312 Tyr Thr Ser Arg Asn Arg Gly Tyr Asn GluAla Pro Ser Val Pro Ala 1090 1095 1100 GAT TAT GCG TCA GTC TAT GAA GAAAAA TCG TAT ACA GAT GGA CGA AGA 3360 Asp Tyr Ala Ser Val Tyr Glu Glu LysSer Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 GAG AAT CCT TGT GAA TTTAAC AGA GGG TAT AGG GAT TAC ACG CCA CTA 3408 Glu Asn Pro Cys Glu Phe AsnArg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 CCA GTT GGT TAT GTGACA AAA GAA TTA GAA TAC TTC CCA GAA ACC GAT 3456 Pro Val Gly Tyr Val ThrLys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 1145 1150 AAG GTA TGG ATTGAG ATT GGA GAA ACG GAA GGA ACA TTT ATC GTG GAC 3504 Lys Val Trp Ile GluIle Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1155 1160 1165 AGC GTG GAATTA CTC CTT ATG GAG GAA TAG 3534 Ser Val Glu Leu Leu Leu Met Glu Glu1170 1175 1177 amino acids amino acid linear protein not provided 28 MetAsp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 7580 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 9095 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr ThrAla 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu LeuSer Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val LeuArg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala AlaThr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly AsnTyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu ArgVal Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln PheArg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala LeuPhe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val SerGln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn PheAsp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser IleArg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile TyrThr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val LeuAsp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro SerAla Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp GluIle Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe SerHis Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser AsnSer Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile HisArg Ser Ala Glu Phe Asn Asn 450 455 460 Ile Ile Ala Ser Asp Ser Ile ThrGln Ile Pro Leu Val Lys Ala His 465 470 475 480 Thr Leu Gln Ser Gly ThrThr Val Val Arg Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu ArgArg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile 500 505 510 Val Asn Ile AsnGly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg 515 520 525 Tyr Ala SerThr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 530 535 540 Arg IlePhe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro 545 550 555 560Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 565 570575 Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser 580585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr595 600 605 Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys AlaVal 610 615 620 Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys ThrAsp Val 625 630 635 640 Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu ValAsp Cys Leu Ser 645 650 655 Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu LeuSer Glu Lys Val Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg AsnLeu Leu Gln Asp Pro Asn 675 680 685 Phe Lys Gly Ile Asn Arg Gln Leu AspArg Gly Trp Arg Gly Ser Thr 690 695 700 Asp Ile Thr Ile Gln Arg Gly AspAsp Val Phe Lys Glu Asn Tyr Val 705 710 715 720 Thr Leu Pro Gly Thr PheAsp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln 725 730 735 Lys Ile Asp Glu SerLys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg 740 745 750 Gly Tyr Ile GluAsp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 755 760 765 Asn Ala LysHis Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780 Pro LeuSer Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg 785 790 795 800Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg 805 810815 Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile 820825 830 Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile835 840 845 Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn LeuGlu 850 855 860 Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala ArgVal Lys 865 870 875 880 Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu LysLeu Glu Trp Glu 885 890 895 Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu SerVal Asp Ala Leu Phe 900 905 910 Val Asn Ser Gln Tyr Asp Gln Leu Gln AlaAsp Thr Asn Ile Ala Met 915 920 925 Ile His Ala Ala Asp Lys Arg Val HisSer Ile Arg Glu Ala Tyr Leu 930 935 940 Pro Glu Leu Ser Val Ile Pro GlyVal Asn Ala Ala Ile Phe Glu Glu 945 950 955 960 Leu Glu Gly Arg Ile PheThr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 965 970 975 Val Ile Lys Asn GlyAsp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val 980 985 990 Lys Gly His ValAsp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu 995 1000 1005 Val ValPro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys 1010 1015 1020Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 10251030 1035 1040 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn ThrAsp Glu 1045 1050 1055 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile TyrPro Asn Asn Thr 1060 1065 1070 Val Thr Cys Asn Asp Tyr Thr Val Asn GlnGlu Glu Tyr Gly Gly Ala 1075 1080 1085 Tyr Thr Ser Arg Asn Arg Gly TyrAsn Glu Ala Pro Ser Val Pro Ala 1090 1095 1100 Asp Tyr Ala Ser Val TyrGlu Glu Lys Ser Tyr Thr Asp Gly Arg Arg 1105 1110 1115 1120 Glu Asn ProCys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu 1125 1130 1135 ProVal Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1140 11451150 Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp1155 1160 1165 Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 3579 basepairs nucleic acid single linear not provided CDS 1..3579 29 ATG GAT AACAAT CCG AAC ATC AAT GAA TGC ATT CCT TAT AAT TGT TTA 48 Met Asp Asn AsnPro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 AGT AAC CCTGAA GTA GAA GTA TTA GGT GGA GAA AGA ATA GAA ACT GGT 96 Ser Asn Pro GluVal Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 TAC ACC CCA ATCGAT ATT TCC TTG TCG CTA ACG CAA TTT CTT TTG AGT 144 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 GAA TTT GTT CCC GGTGCT GGA TTT GTG TTA GGA CTA GTT GAT ATA ATA 192 Glu Phe Val Pro Gly AlaGly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 TGG GGA ATT TTT GGT CCCTCT CAA TGG GAC GCA TTT CTT GTA CAA ATT 240 Trp Gly Ile Phe Gly Pro SerGln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 GAA CAG TTA ATT AAC CAAAGA ATA GAA GAA TTC GCT AGG AAC CAA GCC 288 Glu Gln Leu Ile Asn Gln ArgIle Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 ATT TCT AGA TTA GAA GGA CTAAGC AAT CTT TAT CAA ATT TAC GCA GAA 336 Ile Ser Arg Leu Glu Gly Leu SerAsn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 TCT TTT AGA GAG TGG GAA GCAGAT CCT ACT AAT CCA GCA TTA AGA GAA 384 Ser Phe Arg Glu Trp Glu Ala AspPro Thr Asn Pro Ala Leu Arg Glu 115 120 125 GAG ATG CGT ATT CAA TTC AATGAC ATG AAC AGT GCC CTT ACA ACC GCT 432 Glu Met Arg Ile Gln Phe Asn AspMet Asn Ser Ala Leu Thr Thr Ala 130 135 140 ATT CCT CTT TTT GCA GTT CAAAAT TAT CAA GTT CCT CTT TTA TCA GTA 480 Ile Pro Leu Phe Ala Val Gln AsnTyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 TAT GTT CAA GCT GCA AATTTA CAT TTA TCA GTT TTG AGA GAT GTT TCA 528 Tyr Val Gln Ala Ala Asn LeuHis Leu Ser Val Leu Arg Asp Val Ser 165 170 175 GTG TTT GGA CAA AGG TGGGGA TTT GAT GCC GCG ACT ATC AAT AGT CGT 576 Val Phe Gly Gln Arg Trp GlyPhe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 TAT AAT GAT TTA ACT AGGCTT ATT GGC AAC TAT ACA GAT TAT GCT GTA 624 Tyr Asn Asp Leu Thr Arg LeuIle Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 CGC TGG TAC AAT ACG GGATTA GAA CGT GTA TGG GGA CCG GAT TCT AGA 672 Arg Trp Tyr Asn Thr Gly LeuGlu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 GAT TGG GTA AGG TAT AATCAA TTT AGA AGA GAA TTA ACA CTA ACT GTA 720 Asp Trp Val Arg Tyr Asn GlnPhe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 TTA GAT ATC GTT GCTCTG TTC CCG AAT TAT GAT AGT AGA AGA TAT CCA 768 Leu Asp Ile Val Ala LeuPhe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 ATT CGA ACA GTT TCCCAA TTA ACA AGA GAA ATT TAT ACA AAC CCA GTA 816 Ile Arg Thr Val Ser GlnLeu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 TTA GAA AAT TTT GATGGT AGT TTT CGA GGC TCG GCT CAG GGC ATA GAA 864 Leu Glu Asn Phe Asp GlySer Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 AGA AGT ATT AGG AGTCCA CAT TTG ATG GAT ATA CTT AAC AGT ATA ACC 912 Arg Ser Ile Arg Ser ProHis Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 ATC TAT ACG GAT GCTCAT AGG GGT TAT TAT TAT TGG TCA GGG CAT CAA 960 Ile Tyr Thr Asp Ala HisArg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ATA ATG GCT TCTCCT GTA GGG TTT TCG GGG CCA GAA TTC ACT TTT CCG 1008 Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 CTA TAT GGA ACTATG GGA AAT GCA GCT CCA CAA CAA CGT ATT GTT GCT 1056 Leu Tyr Gly Thr MetGly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 CAA CTA GGT CAGGGC GTG TAT AGA ACA TTA TCG TCC ACT TTA TAT AGA 1104 Gln Leu Gly Gln GlyVal Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 340 345 350 AGA CCT TTT AATATA GGG ATA AAT AAT CAA CAA CTA TCT GTT CTT GAC 1152 Arg Pro Phe Asn IleGly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 GGG ACA GAA TTTGCT TAT GGA ACC TCC TCA AAT TTG CCA TCC GCT GTA 1200 Gly Thr Glu Phe AlaTyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 TAC AGA AAAAGC GGA ACG GTA GAT TCG CTG GAT GAA ATA CCG CCA CAG 1248 Tyr Arg Lys SerGly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 AAT AAC AACGTG CCA CCT AGG CAA GGA TTT AGT CAT CGA TTA AGC CAT 1296 Asn Asn Asn ValPro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 GTT TCA ATGTTT CGT TCA GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA 1344 Val Ser Met PheArg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 AGA GCT CCTATG TTC TCT TGG ATA CAT CGT AGT GCA ACT CTT ACA AAT 1392 Arg Ala Pro MetPhe Ser Trp Ile His Arg Ser Ala Thr Leu Thr Asn 450 455 460 ACA ATT GATCCA GAG AGA ATT AAT CAA ATA CCT TTA GTG AAA GGA TTT 1440 Thr Ile Asp ProGlu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe 465 470 475 480 AGA GTTTGG GGG GGC ACC TCT GTC ATT ACA GGA CCA GGA TTT ACA GGA 1488 Arg Val TrpGly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly 485 490 495 GGG GATATC CTT CGA AGA AAT ACC TTT GGT GAT TTT GTA TCT CTA CAA 1536 Gly Asp IleLeu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln 500 505 510 GTC AATATT AAT TCA CCA ATT ACC CAA AGA TAC CGT TTA AGA TTT CGT 1584 Val Asn IleAsn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg 515 520 525 TAC GCTTCC AGT AGG GAT GCA CGA GTT ATA GTA TTA ACA GGA GCG GCA 1632 Tyr Ala SerSer Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala 530 535 540 TCC ACAGGA GTG GGA GGC CAA GTT AGT GTA AAT ATG CCT CTT CAG AAA 1680 Ser Thr GlyVal Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys 545 550 555 560 ACTATG GAA ATA GGG GAG AAC TTA ACA TCT AGA ACA TTT AGA TAT ACC 1728 Thr MetGlu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr 565 570 575 GATTTT AGT AAT CCT TTT TCA TTT AGA GCT AAT CCA GAT ATA ATT GGG 1776 Asp PheSer Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly 580 585 590 ATAAGT GAA CAA CCT CTA TTT GGT GCA GGT TCT ATT AGT AGC GGT GAA 1824 Ile SerGlu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu 595 600 605 CTTTAT ATA GAT AAA ATT GAA ATT ATT CTA GCA GAT GCA ACA TTT GAA 1872 Leu TyrIle Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 GCAGAA TCT GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCC CTG TTT 1920 Ala GluSer Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe 625 630 635 640ACT TCT TCC AAT CAA ATC GGG TTA AAA ACC GAT GTG ACG GAT TAT CAT 1968 ThrSer Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His 645 650 655ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA TTT TGT 2016 IleAsp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys 660 665 670CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA 2064 LeuAsp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg 675 680 685CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC TTC AGA GGG ATC 2112 LeuSer Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile 690 695 700AAT AGA CAA CCA GAC CGT GGC TGG AGA GGA AGT ACA GAT ATT ACC ATC 2160 AsnArg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile 705 710 715720 CAA GGA GGA GAT GAC GTA TTC AAA GAG AAT TAC GTC ACA CTA CCG GGT 2208Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly 725 730735 ACC GTT GAT GAG TGC TAT CCA ACG TAT TTA TAT CAG AAA ATA GAT GAG 2256Thr Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu 740 745750 TCG AAA TTA AAA GCT TAT ACC CGT TAT GAA TTA AGA GGG TAT ATC GAA 2304Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu 755 760765 GAT AGT CAA GAC TTA GAA ATC TAT TTG ATC CGT TAC AAT GCA AAA CAC 2352Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His 770 775780 GAA ATA GTA AAT GTG CCA GGC ACG GGT TCC TTA TGG CCG CTT TCA GCC 2400Glu Ile Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala 785 790795 800 CAA AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC2448 Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His 805810 815 CTT GAA TGG AAT CCT GAT CTA GAT TGT TCC TGC AGA GAC GGG GAA AAA2496 Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys 820825 830 TGT GCA CAT CAT TCC CAT CAT TTC ACC TTG GAT ATT GAT GTT GGA TGT2544 Cys Ala His His Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys 835840 845 ACA GAC TTA AAT GAG GAC TTA GGT GTA TGG GTG ATA TTC AAG ATT AAG2592 Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys 850855 860 ACG CAA GAT GGC CAT GCA AGA CTA GGG AAT CTA GAG TTT CTC GAA GAG2640 Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu 865870 875 880 AAA CCA TTA TTA GGG GAA GCA CTA GCT CGT GTG AAA AGA GCG GAGAAG 2688 Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys885 890 895 AAG TGG AGA GAC AAA CGA GAG AAA CTG CAG TTG GAA ACA AAT ATTGTT 2736 Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val900 905 910 TAT AAA GAG GCA AAA GAA TCT GTA GAT GCT TTA TTT GTA AAC TCTCAA 2784 Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln915 920 925 TAT GAT AGA TTA CAA GTG GAT ACG AAC ATC GCA ATG ATT CAT GCGGCA 2832 Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala930 935 940 GAT AAA CGC GTT CAT AGA ATC CGG GAA GCG TAT CTG CCA GAG TTGTCT 2880 Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser945 950 955 960 GTG ATT CCA GGT GTC AAT GCG GCC ATT TTC GAA GAA TTA GAGGGA CGT 2928 Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu GlyArg 965 970 975 ATT TTT ACA GCG TAT TCC TTA TAT GAT GCG AGA AAT GTC ATTAAA AAT 2976 Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile LysAsn 980 985 990 GGC GAT TTC AAT AAT GGC TTA TTA TGC TGG AAC GTG AAA GGTCAT GTA 3024 Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly HisVal 995 1000 1005 GAT GTA GAA GAG CAA AAC AAC CAC CGT TCG GTC CTT GTTATC CCA GAA 3072 Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val IlePro Glu 1010 1015 1020 TGG GAG GCA GAA GTG TCA CAA GAG GTT CGT GTC TGTCCA GGT CGT GGC 3120 Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys ProGly Arg Gly 1025 1030 1035 1040 TAT ATC CTT CGT GTC ACA GCA TAT AAA GAGGGA TAT GGA GAG GGC TGC 3168 Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu GlyTyr Gly Glu Gly Cys 1045 1050 1055 GTA ACG ATC CAT GAG ATC GAA GAC AATACA GAC GAA CTG AAA TTC AGC 3216 Val Thr Ile His Glu Ile Glu Asp Asn ThrAsp Glu Leu Lys Phe Ser 1060 1065 1070 AAC TGT GTA GAA GAG GAA GTA TATCCA AAC AAC ACA GTA ACG TGT AAT 3264 Asn Cys Val Glu Glu Glu Val Tyr ProAsn Asn Thr Val Thr Cys Asn 1075 1080 1085 AAT TAT ACT GGG ACT CAA GAAGAA TAT GAG GGT ACG TAC ACT TCT CGT 3312 Asn Tyr Thr Gly Thr Gln Glu GluTyr Glu Gly Thr Tyr Thr Ser Arg 1090 1095 1100 AAT CAA GGA TAT GAC GAAGCC TAT GGT AAT AAC CCT TCC GTA CCA GCT 3360 Asn Gln Gly Tyr Asp Glu AlaTyr Gly Asn Asn Pro Ser Val Pro Ala 1105 1110 1115 1120 GAT TAC GCT TCAGTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA 3408 Asp Tyr Ala Ser ValTyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg 1125 1130 1135 GAG AAT CCTTGT GAA TCT AAC AGA GGC TAT GGG GAT TAC ACA CCA CTA 3456 Glu Asn Pro CysGlu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu 1140 1145 1150 CCG GCTGGT TAT GTA ACA AAG GAT TTA GAG TAC TTC CCA GAG ACC GAT 3504 Pro Ala GlyTyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu Thr Asp 1155 1160 1165 AAGGTA TGG ATT GAG ATC GGA GAA ACA GAA GGA ACA TTC ATC GTG GAT 3552 Lys ValTrp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1170 1175 1180AGC GTG GAA TTA CTC CTT ATG GAG GAA 3579 Ser Val Glu Leu Leu Leu Met GluGlu 1185 1190 1193 amino acids amino acid linear protein not provided 30Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 1015 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 2530 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 4045 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 5560 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 7075 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 8590 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu ArgGlu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu ThrThr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro LeuLeu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser ValLeu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp AlaAla Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile GlyAsn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu GluArg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn GlnPhe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val AlaLeu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr ValSer Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu AsnPhe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg SerIle Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 IleTyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu TyrArg 340 345 350 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser ValLeu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu ProSer Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu AspGlu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly PheSer His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe SerAsn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp IleHis Arg Ser Ala Thr Leu Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg IleAsn Gln Ile Pro Leu Val Lys Gly Phe 465 470 475 480 Arg Val Trp Gly GlyThr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile LeuArg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln 500 505 510 Val Asn IleAsn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg 515 520 525 Tyr AlaSer Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala 530 535 540 SerThr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys 545 550 555560 Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr 565570 575 Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly580 585 590 Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser GlyGlu 595 600 605 Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala ThrPhe Glu 610 615 620 Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val AsnAla Leu Phe 625 630 635 640 Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr AspVal Thr Asp Tyr His 645 650 655 Ile Asp Gln Val Ser Asn Leu Val Asp CysLeu Ser Asp Glu Phe Cys 660 665 670 Leu Asp Glu Lys Arg Glu Leu Ser GluLys Val Lys His Ala Lys Arg 675 680 685 Leu Ser Asp Glu Arg Asn Leu LeuGln Asp Pro Asn Phe Arg Gly Ile 690 695 700 Asn Arg Gln Pro Asp Arg GlyTrp Arg Gly Ser Thr Asp Ile Thr Ile 705 710 715 720 Gln Gly Gly Asp AspVal Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly 725 730 735 Thr Val Asp GluCys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu 740 745 750 Ser Lys LeuLys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu 755 760 765 Asp SerGln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His 770 775 780 GluIle Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala 785 790 795800 Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His 805810 815 Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys820 825 830 Cys Ala His His Ser His His Phe Thr Leu Asp Ile Asp Val GlyCys 835 840 845 Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe LysIle Lys 850 855 860 Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu PheLeu Glu Glu 865 870 875 880 Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg ValLys Arg Ala Glu Lys 885 890 895 Lys Trp Arg Asp Lys Arg Glu Lys Leu GlnLeu Glu Thr Asn Ile Val 900 905 910 Tyr Lys Glu Ala Lys Glu Ser Val AspAla Leu Phe Val Asn Ser Gln 915 920 925 Tyr Asp Arg Leu Gln Val Asp ThrAsn Ile Ala Met Ile His Ala Ala 930 935 940 Asp Lys Arg Val His Arg IleArg Glu Ala Tyr Leu Pro Glu Leu Ser 945 950 955 960 Val Ile Pro Gly ValAsn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg 965 970 975 Ile Phe Thr AlaTyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn 980 985 990 Gly Asp PheAsn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val 995 1000 1005 AspVal Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu 1010 10151020 Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly1025 1030 1035 1040 Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr GlyGlu Gly Cys 1045 1050 1055 Val Thr Ile His Glu Ile Glu Asp Asn Thr AspGlu Leu Lys Phe Ser 1060 1065 1070 Asn Cys Val Glu Glu Glu Val Tyr ProAsn Asn Thr Val Thr Cys Asn 1075 1080 1085 Asn Tyr Thr Gly Thr Gln GluGlu Tyr Glu Gly Thr Tyr Thr Ser Arg 1090 1095 1100 Asn Gln Gly Tyr AspGlu Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala 1105 1110 1115 1120 Asp TyrAla Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg 1125 1130 1135Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu 11401145 1150 Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu ThrAsp 1155 1160 1165 Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr PheIle Val Asp 1170 1175 1180 Ser Val Glu Leu Leu Leu Met Glu Glu 1185 1190

What is claimed is:
 1. An isolated polynucleotide segment comprising asequence region that encodes a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30. 2.The isolated polynucleotide segment of claim 1, comprising a sequenceregion that encodes a polypeptide comprising the amino acid sequence ofSEQ ID NO:10, or SEQ ID NO:12.
 3. The isolated polynucleotide segment ofclaim 1, further defined as DNA.
 4. The polynucleotide of claim 1,comprised within a vector.
 5. The polynucleotide of claim 4, whereinsaid vector is pEG1068, pEG1077, pEG1091, pEG1092, pEG1093, pEG365, orpEG378.
 6. The polynucleotide of claim 4, wherein said polynucleotide isoperatively linked to a promoter, said promoter expressing saidpolynucleotide.
 7. A transformed host cell comprising the polynucleotideof claim
 1. 8. The transformed host cell of claim 7, further defined asa prokaryotic cell.
 9. The transformed host cell of claim 8, furtherdefined as a bacterial cell.
 10. The transformed host cell of claim 9,wherein said bacterial cell is an E. coli, B. thuringiensis, B.subtilis, B. megaterium, or a Pseudomonas spp. cell.
 11. The transformedhost cell of claim 10, wherein said bacterial cell is a B. thuringiensisNRRL B-21579, NRRL B-21580, NRRL B-21581, NRRL B-21635 or NRRL B-21636cell.
 12. The transformed host cell of claim 7, further defined as aneukaryotic cell.
 13. The transformed host cell of claim 12, furtherdefined as a plant cell.
 14. The transformed host cell of claim 13,wherein said plant cell is a corn, wheat, oat, barley, maize, rye, turfgrass, pasture grass, vegetable, berry, fruit, tree, or ornamental plantcell.
 15. The transformed host cell of claim 7, wherein saidpolynucleotide segment is introduced into said cell by means of avector.
 16. The transformed host cell of claim 7, wherein said host cellexpresses said polynucleotide segment to produce an insecticidalpolypeptide.
 17. The isolated polynucleotide segment of claim 1,comprising a sequence region that encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:14 or SEQ ID NO:26.
 18. The isolatedpolynucleotide segment of claim 1, comprising a sequence region thatencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:28or SEQ ID NO:30.